Basic Molecular Genetic Mechanism
Deoxyribonucleic acid (DNA) contains all the information required to build the cells and tissues of an organism
The exact replication of this information in any species assures its genetic continuity from generation to generation and is critical to the normal development of an individual
Information stored in DNA is arranged in hereditary units, now known as genes, that control identifiable traits of an organism. In the process of transcription, the information stored in DNA is copied into ribonucleic acid (RNA), which has three distinct roles in protein synthesis
Messenger RNA (mRNA) carries the instructions from DNA that specify the correct order of amino acids during protein synthesis
Discovery of the structure of DNA in 1953 and subsequent elucidation of how DNA directs the synthesis of RNA
Which then directs the assembly of proteins (the so-called central dogma) were monumental achievements marking the early days of molecular biology
Proteins are largely responsible for regulating gene expression, the entire process whereby the information encoded in DNA is decoded into the proteins that characterize various cell types
DNA and RNA are chemically similar
The primary structures of both are linear polymers composed of monomers called nucleotides
DNA and RNA each consist of only four different nucleotides
In RNA, the sugar is ribose; in DNA, deoxyribose
The nucleotides used in the synthesis of DNA and RNA contain five different bases
The bases adenine (A) and guanine (G) are purines, which contain a pair of fused rings; the bases cytosine (C), thymine (T), and uracil (U) are pyrimidines, which contain a single ring
Both DNA and RNA contain three of these bases A, G, and C
T is found only in DNA
U only in RNA
A single nucleic acid strand has a backbone composed of repeating pentose-phosphate units from which the purine and pyrimidine bases extend as side groups
A nucleic acid strand has an end-to-end chemical orientation: the 5’ end has a hydroxyl or phosphate group on the 5’ carbon of its terminal sugar;
The 3’ end usually has a hydroxyl group on the 3 carbon of its terminal sugar
The chemical linkage between adjacent nucleotides, commonly called a phosphodiester bond actually consists of two phosphodiester bonds, one on the 5’ sides of the phosphate and another on the 3’ side
Polynucleotides can twist and fold into three-dimensional conformations stabilized by noncovalent bonds
DNA and RNA have different three-dimensional conformations
DNA consists of two associated polynucleotide strands that wind together to form a double helix
The two sugar-phosphate backbones are on the outside of the double helix, and the bases project into the interior
The strands are held in the precise register by the formation of base pairs between the two strands: A is paired with T through two hydrogen bonds; G is paired with C through three hydrogen bonds
In natural DNA, A always hydrogen bonds with T and G with C, forming A·T and G·C base pairs
Two polynucleotide strands, or regions thereof, in which all the nucleotides form such base pairs are said to be complementary
But, in theory, and in synthetic DNAs other base pairs can form
The space available in the helix also would allow pairing between the two pyrimidines cytosine and thymine
On the outside of B-form DNA, the spaces between the intertwined strands form two helical grooves of different widths described as the major groove and the minor groove
DNA-binding proteins can “read” the sequence of bases in duplex DNA by contacting atoms in either the major or the minor grooves
By far the most important modifications in the structure of standard B-form DNA come about as a result of protein binding to specific DNA sequences
The unwinding and separation of DNA strands referred to as denaturation, or “melting,” can be induced experimentally by increasing the temperature of a solution of DNA
As the thermal energy increases, the resulting increase in molecular motion eventually breaks the hydrogen bonds and other forces that stabilize the double helix; the strands then separate
Driven apart by the electrostatic repulsion of the negatively charged deoxyribose-phosphate backbone of each strand
The melting temperature Tm at which DNA strands will separate depends on several factors
Molecules that contain a greater proportion of G·C pairs require higher temperatures to denature because the three hydrogen bonds in G·C pairs make these base pairs more stable than A·T pairs, which have only two hydrogen bonds
When the ion concentration is low, this shielding is decreased, increasing the repulsive forces between the strands and reducing the Tm
Agents that destabilize hydrogen bonds, such as formamide or urea, also lower the Tm
Single-stranded DNA molecules that result from denaturation form random coils without an organized structure
Lowering the temperature, increasing the ion concentration, or neutralizing the pH causes the two complementary strands to reassociate into a perfect double helix
DNA partner strands from finding each other and renaturing. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization
A powerful technique used to study the relatedness of two DNA samples and to detect and isolate specific DNA molecules in a mixture containing numerous different DNA sequences
Prokaryotic genomic DNAs and viral DNAs are circular molecules
Circular DNA molecules occur in mitochondria, which are present in almost all eukaryotic cells, and in chloroplasts, which are present in plants and some unicellular eukaryotes
The localized unwinding of a circular DNA molecule (which occurs during DNA replication) induces torsional stress into the remaining portion of the molecule because the ends of the strands are not free to rotate
Resulting in the DNA molecule twisting back on itself, forming super-colls
The enzyme binds to DNA at random sites and breaks a phosphodiester bond in one strand
A one-strand break in DNA is called a nick
Although eukaryotic nuclear DNA is linear, long loops of DNA are fixed in place within chromosomes
The primary structure of RNA is generally similar to that of DNA with two exceptions: the sugar component of RNA, ribose, has a hydroxyl group at the 2 positions, and thymine in DNA is replaced by uracil in RNA
Hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA and provides a chemically reactive group that takes part in RNA-mediated catalysis
Most cellular RNAs are single-stranded and exhibit a variety of conformations
tRNA molecules adopt a well-defined three-dimensional architecture in a solution that is crucial in protein synthesis
The folded domains of RNA molecules not only are structurally analogous to the B helices and strands found in proteins but in some cases also have catalytic capacities
The catalytic RNAs are called ribozymes
The simplest definition of a gene is a “unit of DNA that contains the information to specify synthesis of a single polypeptide chain or functional RNA (such as a tRNA)”
During the synthesis of RNA, the four-base language of DNA containing A, G, C, and T is simply copied, or transcribed, into the four-base language of RNA, which is identical except that U replaces T
During transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA chain
The energetics of the polymerization reaction strongly favors the addition of ribonucleotides to the growing RNA chain
The high-energy bond between the ox and B phosphate of rNTP monomers is replaced by the lower-energy phosphodiester bond between nucleotides
The convention takes place, at the site which RNA polymerase transcription is numbered 1
Downstream denotes the direction in which a template DNA strand is transcribed (or mRNA translated); a downstream sequence is toward the 3 ends relative to the start site, considering the DNA strand with the same polarity as the transcribed RNA
Upstream denotes the opposite direction
Nucleotide positions in the DNA sequence downstream from a start site are indicated by a positive (+) sign; those upstream, by a negative (-) sign
Stages in Transcription: To carry out transcription, RNA polymerase performs several distinct functions, as depicted
During transcription initiation, RNA polymerase recognizes and binds to a specific site, called a promoter
Nuclear RNA polymerases require various protein factors, called general transcription factors
Cellular RNA polymerases melt approximately 14 base pairs of DNA around the transcription start site, which is located on the template strand within the promoter region
Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond
The stage of strand elongation, RNA polymerase moves along the template DNA one base at a time, opening the double-stranded DNA in front of its direction of movement and hybridizing the strands behind it
In the final stage in RNA synthesis, the completed RNA molecule, or primary transcript, is released from the RNA polymerase and the polymerase dissociates from the template DNA
Structure of RNA Polymerases: The RNA polymerases of bacteria, archaea, and eukaryotic cells are fundamentally similar in structure and function
According to a current model of the interaction between bacterial RNA polymerase and promoter DNA, the DNA bends sharply following its entry into the enzyme
The most common arrangement of protein-coding genes in all prokaryotes has a powerful and appealing logic: genes devoted to a single metabolic goal, say, the synthesis of the amino acid tryptophan, is most often found in a contiguous array in the DNA
The arrangement of genes in a functional group is called an operon
Each section of the mRNA represents the unit (or gene) that encodes one of the proteins in the series
In prokaryotic DNA the genes are closely packed with very few noncoding gaps, and the DNA is transcribed directly into colinear mRNA, which then is translated into protein
Concluded that the eukaryotic gene existed in pieces of a coding sequence, the exons, separated by non-protein-coding segments, the introns
Introns are present in the DNA of viruses that infect eukaryotic cells.
Prokaryotic cells, which have no nuclei
Translation of an mRNA into protein can begin from the 5’ end of the mRNA even while the 3’ end is still being synthesized by RNA polymerase
All eukaryotic pre-mRNAs initially are modified at the two ends, and these modifications are retained in mRNAs
As the 5’ end of a nascent RNA chain emerges from the surface of RNA polymerase II, it is immediately acted on by several enzymes that together synthesize the 5’ cap
A 7-methylguanylate that is connected to the terminal nucleotide of the RNA by an unusual 5’,5’ triphosphate linkage
Processing at the 3’ end of a pre-mRNA involves cleavage by an endonuclease to yield a free 3-hydroxyl group to which a string of adenylic acid residues is added one at a time by an enzyme called poly(A) polymerase
The final step in the processing of many different eukaryotic mRNA molecules is RNA splicing: the internal cleavage of a transcript to excise the introns, followed by ligation of the coding exons
Prokaryotic mRNAs also usually have 5’ and 3’ UTRs, but these are much shorter than those in eukaryotic mRNAs, generally containing fewer than 10 nucleotides
In comparison to bacterial and archaeal genes, the vast majority of genes in higher, multicellular eukaryotes contain multiple introns
Individual repeated protein domains often are encoded by one exon or a small number of exons that code for identical or nearly identical amino acid sequences
The presence of multiple introns in many eukaryotic genes permits the expression of multiple, related proteins from a single gene by means of alternative splicing
Fibronectin, a multidomain extracellular adhesive protein found in mammals, provides a good example of alternative splicing
During the formation of blood clots, however, the fibrin-binding domains of hepatocyte fibronectin bind to fibrin, one of the principal constituents of clots
More than 20 different isoforms of fibronectin have been identified, each encoded by a different
Alternatively spliced mRNA composed of a unique combination of fibronectin gene exons
The structure and function of a cell are determined by the proteins it contains, the control of gene expression is a fundamental aspect of molecular cell biology
Most commonly, the “decision” to initiate transcription of the gene encoding a particular protein is the major mechanism for controlling the production of the encoded protein in a cell
When transcription of a gene is repressed, the corresponding mRNA and encoded protein or proteins are synthesized at low rates
In most bacteria and other single-celled organisms, gene expression is highly regulated in order to adjust the cell’s enzymatic machinery and structural components to changes in the nutritional and physical environment
Multicellular organisms, control of gene expression is largely directed toward assuring that the right gene is expressed in the right cell at the right time during embryological development and tissue differentiation
In E. coli, about half the genes are clustered into operons each of which encodes enzymes involved in a particular metabolic pathway or proteins that interact to form one multisubunit protein
Transcription of operons, as well as of isolated genes, is controlled by an interplay between RNA polymerase and specific repressor and activator proteins
When E. coli is in an environment that lacks lactose, synthesis of lac mRNA is repressed, so that cellular energy is not wasted synthesizing enzymes the cells cannot use
In an environment that has both lactose and glucose, E. coli cells usually metabolize glucose, the central molecule of carbohydrate metabolism
Transcription of the lac operon under different conditions are controlled by lac repressor and catabolite activator protein (CAP)
Each of which binds to a specific DNA sequence in the lac transcription-control region
When no lactose is present, binding of the lac repressor to a sequence called the lac operator
Which overlaps the transcription start site, blocks transcription initiation by the polymerase
Once glucose is depleted from the media and the intracellular glucose concentration falls, E. coli cells respond by synthesizing cyclic AMP, cAMP
The promoters for different E. coli genes exhibit considerable homology, their exact sequences differ
Promoters that support a high rate of transcription initiation are called strong promoters, those that support a low rate of transcription initiation are called weak promoters
Transcription of most E. coli genes is regulated by processes similar to those described for the lac operon
The general mechanism involves a specific repressor that binds to the operator region of a gene or operon, thereby blocking transcription initiation
A small molecule (or molecules), called an inducer, binds to the repressor
Controlling its DNA-binding activity and consequently the rate of transcription as appropriate for the needs of the cell
Specific activator proteins, such as CAP in the lac operon, also control transcription of some but not all bacterial genes
These activators bind to DNA together with the RNA polymerase, stimulating transcription from a specific promoter
Most E. coli promoters interact with o70 -RNA polymerase, the major form of the bacterial enzyme
Transcription of certain groups of genes is carried out by E. coli RNA polymerases containing one of several alternative sigma factors that recognize different consensus promoter sequences than o70 does
The sequence of one E. coli sigma factor, o54, is distinctly different from that of all the o70-like factors.
Transcription of genes by RNA polymerases containing o54 is regulated by activators whose binding sites in DNA, referred to as enhancers
Generally are located 80–160 base pairs upstream from the start site
The best-characterized o54 -activator—the NtrC protein (nitrogen regulatory protein C)
Stimulates transcription from the promoter of the glnA gene
Large protein pores in the E. coli outer membrane allow ions to diffuse freely between the external environment and the periplasmic space
When the phosphate concentration in the environment falls, it also falls in the periplasmic space, causing phosphate to dissociate from the PhoR periplasmic domain
Many other bacterial responses are regulated by two proteins with homology to PhoR and PhoB
The first protein, called a sensor, contains a transmitter domain homologous to the PhoR protein kinase domain
The second protein, called a response regulator, contains a receiver domain homologous to the region of PhoB that is phosphorylated by activated PhoR
The translation is the whole process by which the nucleotide sequence of an mRNA is used to order and to join the amino acids in a polypeptide chain
Messenger RNA (mRNA)
Carries the genetic information transcribed from DNA in the form of a series of three-nucleotide sequences - called codons (each specifies a particular amino acid.
Transfer RNA (tRNA)
Very important to decipher the codons in mRNA. Each type of amino acid has its own subset of tRNAs, which bind the amino acid and carry it to the growing end of a polypeptide chain if the next codon in the mRNA calls for it.
Ribosomal RNA (rRNA)
Associates with a set of proteins to create ribosomes. These structures (physically) move along an mRNA molecule, catalyze the assembly of amino acids into polypeptide chains. They bind tRNAs and various other accessory proteins necessary for protein synthesis.
These types of RNA participate in translation in all cells
The development of three functionally distinct RNAs was probably the molecular key to the origin of life
The genetic code used by cells is a triplet code, with every three-nucleotide sequence, or codon, being “read” from a specified starting point in the mRNA
A total of 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop codons
The different codons for a given amino acid are said to be synonymous
Synthesis of all polypeptide chains in prokaryotic and eukaryotic cells begins with the amino acid methionine
In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionally is used as an initiator codon for methionine in eukaryotes
The three codons UAA, UGA, and UAG do not specify amino acids but constitute stop (termination) codons that mark the carboxyl terminus of polypeptide chains in almost all cells
The sequence of codons that runs from a specific start codon to a stop codon is called a reading frame
The linear array of ribonucleotides in groups of three in mRNA specifies the precise linear sequence of amino acids in a polypeptide chain
Particular mRNA theoretically could be translated in three different reading frames
The meaning of each codon is the same in most known organisms (a strong argument that life on earth evolved only once)
The genetic code has been found to differ for a few codons in many mitochondria, in ciliated protozoans, and in Acetabularia, a single-celled plant
Translation/decoding of the four nucleotide language of DNA and mRNA into the 20 amino acid language of proteins requires tRNA and enzymes called aminoacyl-tRNA synthetases
tRNA molecule must become chemically linked to a particular amino acid via a high-energy bond, forming an aminoacyl-tRNA; the anticodon in the tRNA then base-pairs with a codon in mRNA so that the activated amino acid can be added to the growing polypeptide chain to participate in protein synthesis
Some 30–40 different tRNAs have been identified in bacterial cells and as many as 50–100 in animal and plant cells
The function of tRNA molecules, which are 70–80 nucleotides long, depends on their precise three-dimensional structures
The four stems are short double helices stabilized by Watson-Crick base pairing; three of the four stems have loops containing seven or eight bases at their ends
While the remaining, unlooped stem contains the free 3 and 5 ends of the chain
The first and second bases of a codon almost always form standard Watson-Crick base pairs with the third and second bases, of the corresponding anticodon
But four nonstandard interactions can occur between bases in the wobble position
Adenine rarely is found in the anticodon wobble position, many tRNAs in plants and animals contain inosine (I), a deaminated product of adenine, at this position
Inosine can form nonstandard base pairs with A, C, and U
A tRNA with inosine in the wobble position thus can recognize the corresponding mRNA codons with A, C, or U in the third (wobble) position
The first step in decoding the genetic message: attachment of the appropriate amino acid to a tRNA, is catalyzed by a specific aminoacyl-tRNA synthetase
Second step: Recognition of the codon or codons specifying a given amino acid by a particular tRNA
Each of the 20 different synthetases recognizes one amino acid and all its compatible, or cognate, tRNAs
Some amino acids are so similar structurally, aminoacyl-tRNA synthetases sometimes make mistakes
Can be corrected but, by the enzymes themselves which have a proofreading activity that checks the fit in their amino acid binding pocket
Of the many components that participate in translating mRNA had to interact in free solution
The likelihood of simultaneous collisions occurring would be really low
Small proteins of 100–200 amino acids are therefore made in a minute or less
However, it takes 2–3 hours to make the largest known protein (titin) which is found in muscle and contains about 30,000 amino acid residues
The electron microscope helped ribosomes to first be discovered as small, discrete, RNA-rich particles in cells that secrete large amounts of protein
A ribosome is composed of three (in bacteria) or four (in eukaryotes) different rRNA molecules and as many as 83 proteins, organized into a large subunit and a small subunit
The small ribosomal subunit contains a single rRNA molecule, referred to as small rRNA
The large subunit contains a molecule of large rRNA and one molecule of 5S rRNA, plus an additional molecule of 5.8S rRNA invertebrates
The sequences of the small and large rRNAs from several thousand organisms are now known
Although the primary nucleotide sequences of these rRNAs vary considerably, the same parts of each type of rRNA theoretically can form base-paired stem-loops
Which would generate a similar three-dimensional structure for each rRNA in all organisms
During translation, a ribosome moves along an mRNA chain, interacting with various protein factors and tRNAs and very likely undergoing large conformational changes
The AUG codon for methionine functions as the start codon in the vast majority of mRNAs
A critical aspect of translation initiation is to begin protein synthesis at the start codon, thereby establishing the correct reading frame for the entire mRNA
Both prokaryotes and eukaryotes contain two different methionine tRNAs: tRNAiMet can initiate protein synthesis, and tRNAMet can incorporate methionine only into a growing protein chain
The first stage of translation: a ribosome assembles, complexed with an mRNA and an activated initiator tRNA
Which is correctly positioned at the start codon
A translation preinitiation complex is formed when the 40S subunit–eIF3 complex is bound by eIF1A and a ternary complex of the Met-tRNAiMet, eIF2, and GTP
Cells can regulate protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP
The phosphorylated complex is unable to exchange the bound GDP for GTP and cannot bind Met-tRNAiMet, inhibiting protein synthesis
The mRNA-eIF4 complex then associates with the preinitiation complex through an interaction of the eIF4G subunit and eIF3, forming the initiation complex
Scanning stops when the tRNAiMet anticodon recognizes the start codon
Which is the first AUG downstream from the 5’ end in most eukaryotic mRNAs
Recognition of the start codon leads to hydrolysis of the GTP associated with eIF2, an irreversible step that prevents further scanning
Once the small ribosomal subunit with its bound Met-tRNAiMet is correctly positioned at the start codon, union with the large (60S) ribosomal subunit completes the formation of an 80S ribosome
This requires the action of another factor (eIF5) and hydrolysis of a GTP associated with it
The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5 capped end as just described
But, some cellular mRNAs contain an internal ribosome entry site (IRES) located far downstream of the 5’ end
The correctly positioned eukaryotic 80S ribosome–Met-tRNAiMet complex is now ready to begin the task of stepwise addition of amino acids by the in-frame translation of the
mRNA
Special proteins called elongation factors (EFs) require to carry out the process of chain elongation
The key steps in elongation are the entry of each succeeding aminoacyl-tRNA, formation of a peptide bond, and the movement, or translocation, of the ribosome one codon at a time along the mRNA
The completion of translation initiation, as noted already, Met-tRNAiMet is bound to the P site on the assembled 80S ribosome
This region is called the P site - because the tRNA is chemically liked to the growing polypeptide chain
The second (aminoacyl-tRNA) is brought into the ribosome as a ternary
complex in association with EF1 ox- GTP and becomes bound to the A site, so named because it is where aminoacylated tRNAs bind
The hydrolysis of GTP promotes a conformational change in the ribosome that leads to tight binding of the aminoacyl-tRNA in the A site and release of the resulting EF1ox-GDP complex
With the initiating Met-tRNAiMet at the P site and the second aminoacyl-tRNA tightly bound at the A site
The amino group of the second amino acid reacts with the “activated” (ester-linked) methionine on the initiator tRNA, forming a peptide bond
Following peptide bond synthesis, the ribosome is translocated along the mRNA at a distance equal to one codon
This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2ox-GTP
As a result of translocation, tRNAiMet, now without its activated methionine, is moved to the E (exit) site on the ribosome; concurrently
The second tRNA, now covalently bound to a dipeptide (a peptidyl-tRNA), is moved to the P site
The locations of tRNAs bound at the A, P, and E sites are visible in the recently determined crystal structure of the bacterial ribosome
Base pairing is apparent between the tRNAs in the A and P sites with their respective codons in mRNA
The final stage of translation, like initiation and elongation, requires highly specific molecular signals that decide the fate of the mRNA–ribosome–tRNA-peptidyl complex
Two types of specific protein release factors (RFs) have been discovered
Eukaryotic eRF1, whose shape is similar to that of tRNAs, apparently acts by binding to the ribosomal A site and recognizing stop codons directly
The second eukaryotic release factor, eRF3, is a GTP-binding protein
After its release from the ribosome, a newly synthesized protein folds into its native three-dimensional conformation, a process facilitated by other proteins called chaperones
Hydrolysis of the bound GTP is thought to cause conformational changes in the GTPase itself or other associated proteins that are critical to various complex molecular processes
The translation of a single eukaryotic mRNA molecule to yield a typical-sized protein takes 30–60 seconds
Two phenomena significantly increase the overall rate at which cells can synthesize a protein:
The simultaneous translation of a single mRNA molecule by multiple ribosomes and rapid recycling of ribosomal subunits after they disengage from the 3’ end of an mRNA
Simultaneous translation of an mRNA by multiple ribosomes is readily observable in electron micrographs and by sedimentation analysis, revealing mRNA attached to multiple ribosomes bearing nascent growing polypeptide chains
Structures that are referred to as polyribosomes (or polysomes) were seen to be circular in electron micrographs of some tissues
Multiple copies of a cytosolic protein are found in all eukaryotic cells
Analogous to RNA, DNA is synthesized from deoxynucleoside 5’-triphosphate precursors (dNTPs)
DNA synthesis always proceeds in the 5’ → 3’ direction
Because chain growth results from the formation of a phosphodiester bond between the 3 oxygen of a growing strand and the phosphate of a dNTP
DNA polymerases can’t initiate chain synthesis de novo; instead, they require a short, preexisting RNA or DNA strand, called a primer, to begin chain growth
For DNA to function properly as a template during replication the two intertwined strands must be unwound or melted
To make the bases available for base pairing with the bases of the dNTPs that are polymerized into the newly synthesized daughter strands
The DNA region at which all these proteins come together to carry out the synthesis of daughter strands is called the replication fork, or growing fork
Synthesis of one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5’→3’ direction
The same direction as the movement of the replication fork (or growing fork)
There is a problem that comes in the synthesis of the other daughter strand
Called lagging strand
Eukaryotic proteins that participate in DNA replication has come largely from studies with small viral DNAs, particularly SV40 DNA, the circular genome of a small virus that infects monkeys
All other proteins involved in SV40 DNA replication are provided by the host cell
After parental DNA is separated into single-stranded templates at the replication fork, it is bound by multiple copies of RPA (replication protein A), a heterotrimeric protein
Both parental DNA strands that are exposed by local unwinding at a replication fork are copied into a daughter strand
The general consensus is that all prokaryotic and eukaryotic cells employ a bidirectional mechanism of DNA replication
Taking SV40 DNA for example, replication is initiated by binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins to form two replication forks
Activation of MCM helicase activity, which is required to initiate cellular DNA replication, is regulated by specific protein kinases called S-phase cyclin-dependent kinases
Viruses cannot reproduce by themselves and must commandeer a host cell’s machinery to synthesize viral proteins and in some cases to replicate the viral genome
RNA viruses, which usually replicate in the host-cell cytoplasm, have an RNA genome, and DNA viruses, which commonly replicate in the host-cell nucleus, have a DNA genome
Viral genomes may be single- or double-stranded, depending on the specific type of virus
The entire infectious virus particle, called a virion, consists of nucleic acid and an outer shell of protein
The surface of a virion contains many copies of one type of protein that binds specifically to multiple copies of a receptor protein on a host cell
A virus that infects only bacteria is called a bacteriophage, or simply a phage
The nucleic acid of a virion is enclosed within a protein coat, or capsid, composed of multiple copies of one protein or a few different proteins
Each of which is encoded by a single viral gene
A capsid plus the enclosed nucleic acid is called a nucleocapsid
Nature has found two basic ways of arranging the multiple capsid protein subunits and the viral genome into a nucleocapsid
In some viruses, multiple copies of a single coat protein form a helical structure that encloses and protects the viral RNA or DNA
Which runs in a helical groove within the protein tube
The number and arrangement of coat proteins in icosahedral, or quasi-spherical, viruses differ somewhat depending on their size
In large quasi-spherical viruses, each face of the icosahedron is composed of more than three subunits
In the smaller viruses (e.g.poliovirus), clefts that encircle each of the vertices of the icosahedral structure interact with receptors on the surface of host cells during infection
In the larger viruses (e.g., adenovirus), long fiber-like proteins extending from the nucleocapsid interact with cell-surface receptors on host cells
Many DNA bacteriophages, the viral DNA is located within an icosahedral “head” that is attached to a rodlike “tail”
During infection, viral proteins at the tip of the tail bind to host-cell receptors, and then the viral DNA passes down the tail into the cytoplasm of the host cell
The number of infectious viral particles in a sample can be quantified by a plaque assay
This assay is performed by culturing a dilute sample of viral particles on a plate covered with host cells and then counting the number of local lesions, called plaques, that develop
Since all the progeny virions in a plaque are derived from a single parental virus, they constitute a virus clone
This type of plaque assay is in standard use for bacterial and animal viruses
Plant viruses can be assayed similarly by counting local lesions on plant leaves inoculated with viruses
The lytic cycle of growth proceeds through the following general stages
1. Adsorption—Virion interacts with a host cell by binding multiple copies of capsid protein to specific receptors on the cell surface.
2. Penetration—Viral genome crosses the plasma membrane. For animal and plant viruses, viral proteins also enter the host cell.
3. Replication—Viral mRNAs are produced with the aid of the host-cell transcription machinery (DNA viruses) or by viral enzymes (RNA viruses). For both types of viruses, viral mRNAs are translated by the host cell translation machinery. Production of multiple copies of the viral genome is carried out either by viral proteins alone or with the help of host-cell proteins
4. Assembly—Viral proteins and replicated genomes associate to form progeny virions.
5. Release—Infected cells either ruptures suddenly (lysis), releasing all the newly formed virions at once, or disintegrates gradually, with a slow release of virions
Viral capsid proteins generally are made in large amounts because many copies of them are required for the assembly of each progeny virion
In each infected cell, about 100–200 T4 progeny virions are produced and released by lysis
In most such viruses, the DNA genome is transported (with some associated proteins) into the cell nucleus
Once inside the nucleus, the viral DNA is transcribed into RNA by the host’s transcription machinery
Most plant and animal viruses with an RNA genome do not require nuclear functions for lytic replication
In some of these viruses, a virus-encoded enzyme that enters the host during penetration transcribes the genomic RNA into mRNAs in the cell cytoplasm
mRNA is directly translated into viral proteins by the host cell translation machinery
One or more of these proteins then produces additional copies of the viral RNA genome
Lastly, progeny genomes are assembled with newly synthesized capsid proteins into progeny virions in the cytoplasm
After the synthesis of hundreds to thousands of new virions has been completed, most infected bacterial cells and some infected plant and animal cells are lysed, releasing all the virions at once
Some bacterial viruses, called temperate phages, can establish a nonlytic association with their host cells that do not kill the cell
The integrated viral DNA, called a prophage, is replicated as part of the cell’s DNA from one host-cell generation to the next
This phenomenon is referred to as lysogeny
The genomes of a number of animal viruses also can integrate into the host cell genome
The most important are the retroviruses, which are enveloped viruses with a genome consisting of two identical strands of RNA
In the retroviral life cycle, a viral enzyme called reverse transcriptase initially copies the viral RNA genome into single-stranded DNA complementary to the virion RNA; the same enzyme then catalyzes the synthesis of a complementary DNA strand
The resulting double-stranded DNA is integrated into the chromosomal DNA of the infected cell
The integrated DNA is called a provirus
Some retroviruses contain cancer-causing genes (oncogenes), and cells infected by such retroviruses are oncogenically transformed into tumour cells
Deoxyribonucleic acid (DNA) contains all the information required to build the cells and tissues of an organism
The exact replication of this information in any species assures its genetic continuity from generation to generation and is critical to the normal development of an individual
Information stored in DNA is arranged in hereditary units, now known as genes, that control identifiable traits of an organism. In the process of transcription, the information stored in DNA is copied into ribonucleic acid (RNA), which has three distinct roles in protein synthesis
Messenger RNA (mRNA) carries the instructions from DNA that specify the correct order of amino acids during protein synthesis
Discovery of the structure of DNA in 1953 and subsequent elucidation of how DNA directs the synthesis of RNA
Which then directs the assembly of proteins (the so-called central dogma) were monumental achievements marking the early days of molecular biology
Proteins are largely responsible for regulating gene expression, the entire process whereby the information encoded in DNA is decoded into the proteins that characterize various cell types
DNA and RNA are chemically similar
The primary structures of both are linear polymers composed of monomers called nucleotides
DNA and RNA each consist of only four different nucleotides
In RNA, the sugar is ribose; in DNA, deoxyribose
The nucleotides used in the synthesis of DNA and RNA contain five different bases
The bases adenine (A) and guanine (G) are purines, which contain a pair of fused rings; the bases cytosine (C), thymine (T), and uracil (U) are pyrimidines, which contain a single ring
Both DNA and RNA contain three of these bases A, G, and C
T is found only in DNA
U only in RNA
A single nucleic acid strand has a backbone composed of repeating pentose-phosphate units from which the purine and pyrimidine bases extend as side groups
A nucleic acid strand has an end-to-end chemical orientation: the 5’ end has a hydroxyl or phosphate group on the 5’ carbon of its terminal sugar;
The 3’ end usually has a hydroxyl group on the 3 carbon of its terminal sugar
The chemical linkage between adjacent nucleotides, commonly called a phosphodiester bond actually consists of two phosphodiester bonds, one on the 5’ sides of the phosphate and another on the 3’ side
Polynucleotides can twist and fold into three-dimensional conformations stabilized by noncovalent bonds
DNA and RNA have different three-dimensional conformations
DNA consists of two associated polynucleotide strands that wind together to form a double helix
The two sugar-phosphate backbones are on the outside of the double helix, and the bases project into the interior
The strands are held in the precise register by the formation of base pairs between the two strands: A is paired with T through two hydrogen bonds; G is paired with C through three hydrogen bonds
In natural DNA, A always hydrogen bonds with T and G with C, forming A·T and G·C base pairs
Two polynucleotide strands, or regions thereof, in which all the nucleotides form such base pairs are said to be complementary
But, in theory, and in synthetic DNAs other base pairs can form
The space available in the helix also would allow pairing between the two pyrimidines cytosine and thymine
On the outside of B-form DNA, the spaces between the intertwined strands form two helical grooves of different widths described as the major groove and the minor groove
DNA-binding proteins can “read” the sequence of bases in duplex DNA by contacting atoms in either the major or the minor grooves
By far the most important modifications in the structure of standard B-form DNA come about as a result of protein binding to specific DNA sequences
The unwinding and separation of DNA strands referred to as denaturation, or “melting,” can be induced experimentally by increasing the temperature of a solution of DNA
As the thermal energy increases, the resulting increase in molecular motion eventually breaks the hydrogen bonds and other forces that stabilize the double helix; the strands then separate
Driven apart by the electrostatic repulsion of the negatively charged deoxyribose-phosphate backbone of each strand
The melting temperature Tm at which DNA strands will separate depends on several factors
Molecules that contain a greater proportion of G·C pairs require higher temperatures to denature because the three hydrogen bonds in G·C pairs make these base pairs more stable than A·T pairs, which have only two hydrogen bonds
When the ion concentration is low, this shielding is decreased, increasing the repulsive forces between the strands and reducing the Tm
Agents that destabilize hydrogen bonds, such as formamide or urea, also lower the Tm
Single-stranded DNA molecules that result from denaturation form random coils without an organized structure
Lowering the temperature, increasing the ion concentration, or neutralizing the pH causes the two complementary strands to reassociate into a perfect double helix
DNA partner strands from finding each other and renaturing. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization
A powerful technique used to study the relatedness of two DNA samples and to detect and isolate specific DNA molecules in a mixture containing numerous different DNA sequences
Prokaryotic genomic DNAs and viral DNAs are circular molecules
Circular DNA molecules occur in mitochondria, which are present in almost all eukaryotic cells, and in chloroplasts, which are present in plants and some unicellular eukaryotes
The localized unwinding of a circular DNA molecule (which occurs during DNA replication) induces torsional stress into the remaining portion of the molecule because the ends of the strands are not free to rotate
Resulting in the DNA molecule twisting back on itself, forming super-colls
The enzyme binds to DNA at random sites and breaks a phosphodiester bond in one strand
A one-strand break in DNA is called a nick
Although eukaryotic nuclear DNA is linear, long loops of DNA are fixed in place within chromosomes
The primary structure of RNA is generally similar to that of DNA with two exceptions: the sugar component of RNA, ribose, has a hydroxyl group at the 2 positions, and thymine in DNA is replaced by uracil in RNA
Hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA and provides a chemically reactive group that takes part in RNA-mediated catalysis
Most cellular RNAs are single-stranded and exhibit a variety of conformations
tRNA molecules adopt a well-defined three-dimensional architecture in a solution that is crucial in protein synthesis
The folded domains of RNA molecules not only are structurally analogous to the B helices and strands found in proteins but in some cases also have catalytic capacities
The catalytic RNAs are called ribozymes
The simplest definition of a gene is a “unit of DNA that contains the information to specify synthesis of a single polypeptide chain or functional RNA (such as a tRNA)”
During the synthesis of RNA, the four-base language of DNA containing A, G, C, and T is simply copied, or transcribed, into the four-base language of RNA, which is identical except that U replaces T
During transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA chain
The energetics of the polymerization reaction strongly favors the addition of ribonucleotides to the growing RNA chain
The high-energy bond between the ox and B phosphate of rNTP monomers is replaced by the lower-energy phosphodiester bond between nucleotides
The convention takes place, at the site which RNA polymerase transcription is numbered 1
Downstream denotes the direction in which a template DNA strand is transcribed (or mRNA translated); a downstream sequence is toward the 3 ends relative to the start site, considering the DNA strand with the same polarity as the transcribed RNA
Upstream denotes the opposite direction
Nucleotide positions in the DNA sequence downstream from a start site are indicated by a positive (+) sign; those upstream, by a negative (-) sign
Stages in Transcription: To carry out transcription, RNA polymerase performs several distinct functions, as depicted
During transcription initiation, RNA polymerase recognizes and binds to a specific site, called a promoter
Nuclear RNA polymerases require various protein factors, called general transcription factors
Cellular RNA polymerases melt approximately 14 base pairs of DNA around the transcription start site, which is located on the template strand within the promoter region
Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond
The stage of strand elongation, RNA polymerase moves along the template DNA one base at a time, opening the double-stranded DNA in front of its direction of movement and hybridizing the strands behind it
In the final stage in RNA synthesis, the completed RNA molecule, or primary transcript, is released from the RNA polymerase and the polymerase dissociates from the template DNA
Structure of RNA Polymerases: The RNA polymerases of bacteria, archaea, and eukaryotic cells are fundamentally similar in structure and function
According to a current model of the interaction between bacterial RNA polymerase and promoter DNA, the DNA bends sharply following its entry into the enzyme
The most common arrangement of protein-coding genes in all prokaryotes has a powerful and appealing logic: genes devoted to a single metabolic goal, say, the synthesis of the amino acid tryptophan, is most often found in a contiguous array in the DNA
The arrangement of genes in a functional group is called an operon
Each section of the mRNA represents the unit (or gene) that encodes one of the proteins in the series
In prokaryotic DNA the genes are closely packed with very few noncoding gaps, and the DNA is transcribed directly into colinear mRNA, which then is translated into protein
Concluded that the eukaryotic gene existed in pieces of a coding sequence, the exons, separated by non-protein-coding segments, the introns
Introns are present in the DNA of viruses that infect eukaryotic cells.
Prokaryotic cells, which have no nuclei
Translation of an mRNA into protein can begin from the 5’ end of the mRNA even while the 3’ end is still being synthesized by RNA polymerase
All eukaryotic pre-mRNAs initially are modified at the two ends, and these modifications are retained in mRNAs
As the 5’ end of a nascent RNA chain emerges from the surface of RNA polymerase II, it is immediately acted on by several enzymes that together synthesize the 5’ cap
A 7-methylguanylate that is connected to the terminal nucleotide of the RNA by an unusual 5’,5’ triphosphate linkage
Processing at the 3’ end of a pre-mRNA involves cleavage by an endonuclease to yield a free 3-hydroxyl group to which a string of adenylic acid residues is added one at a time by an enzyme called poly(A) polymerase
The final step in the processing of many different eukaryotic mRNA molecules is RNA splicing: the internal cleavage of a transcript to excise the introns, followed by ligation of the coding exons
Prokaryotic mRNAs also usually have 5’ and 3’ UTRs, but these are much shorter than those in eukaryotic mRNAs, generally containing fewer than 10 nucleotides
In comparison to bacterial and archaeal genes, the vast majority of genes in higher, multicellular eukaryotes contain multiple introns
Individual repeated protein domains often are encoded by one exon or a small number of exons that code for identical or nearly identical amino acid sequences
The presence of multiple introns in many eukaryotic genes permits the expression of multiple, related proteins from a single gene by means of alternative splicing
Fibronectin, a multidomain extracellular adhesive protein found in mammals, provides a good example of alternative splicing
During the formation of blood clots, however, the fibrin-binding domains of hepatocyte fibronectin bind to fibrin, one of the principal constituents of clots
More than 20 different isoforms of fibronectin have been identified, each encoded by a different
Alternatively spliced mRNA composed of a unique combination of fibronectin gene exons
The structure and function of a cell are determined by the proteins it contains, the control of gene expression is a fundamental aspect of molecular cell biology
Most commonly, the “decision” to initiate transcription of the gene encoding a particular protein is the major mechanism for controlling the production of the encoded protein in a cell
When transcription of a gene is repressed, the corresponding mRNA and encoded protein or proteins are synthesized at low rates
In most bacteria and other single-celled organisms, gene expression is highly regulated in order to adjust the cell’s enzymatic machinery and structural components to changes in the nutritional and physical environment
Multicellular organisms, control of gene expression is largely directed toward assuring that the right gene is expressed in the right cell at the right time during embryological development and tissue differentiation
In E. coli, about half the genes are clustered into operons each of which encodes enzymes involved in a particular metabolic pathway or proteins that interact to form one multisubunit protein
Transcription of operons, as well as of isolated genes, is controlled by an interplay between RNA polymerase and specific repressor and activator proteins
When E. coli is in an environment that lacks lactose, synthesis of lac mRNA is repressed, so that cellular energy is not wasted synthesizing enzymes the cells cannot use
In an environment that has both lactose and glucose, E. coli cells usually metabolize glucose, the central molecule of carbohydrate metabolism
Transcription of the lac operon under different conditions are controlled by lac repressor and catabolite activator protein (CAP)
Each of which binds to a specific DNA sequence in the lac transcription-control region
When no lactose is present, binding of the lac repressor to a sequence called the lac operator
Which overlaps the transcription start site, blocks transcription initiation by the polymerase
Once glucose is depleted from the media and the intracellular glucose concentration falls, E. coli cells respond by synthesizing cyclic AMP, cAMP
The promoters for different E. coli genes exhibit considerable homology, their exact sequences differ
Promoters that support a high rate of transcription initiation are called strong promoters, those that support a low rate of transcription initiation are called weak promoters
Transcription of most E. coli genes is regulated by processes similar to those described for the lac operon
The general mechanism involves a specific repressor that binds to the operator region of a gene or operon, thereby blocking transcription initiation
A small molecule (or molecules), called an inducer, binds to the repressor
Controlling its DNA-binding activity and consequently the rate of transcription as appropriate for the needs of the cell
Specific activator proteins, such as CAP in the lac operon, also control transcription of some but not all bacterial genes
These activators bind to DNA together with the RNA polymerase, stimulating transcription from a specific promoter
Most E. coli promoters interact with o70 -RNA polymerase, the major form of the bacterial enzyme
Transcription of certain groups of genes is carried out by E. coli RNA polymerases containing one of several alternative sigma factors that recognize different consensus promoter sequences than o70 does
The sequence of one E. coli sigma factor, o54, is distinctly different from that of all the o70-like factors.
Transcription of genes by RNA polymerases containing o54 is regulated by activators whose binding sites in DNA, referred to as enhancers
Generally are located 80–160 base pairs upstream from the start site
The best-characterized o54 -activator—the NtrC protein (nitrogen regulatory protein C)
Stimulates transcription from the promoter of the glnA gene
Large protein pores in the E. coli outer membrane allow ions to diffuse freely between the external environment and the periplasmic space
When the phosphate concentration in the environment falls, it also falls in the periplasmic space, causing phosphate to dissociate from the PhoR periplasmic domain
Many other bacterial responses are regulated by two proteins with homology to PhoR and PhoB
The first protein, called a sensor, contains a transmitter domain homologous to the PhoR protein kinase domain
The second protein, called a response regulator, contains a receiver domain homologous to the region of PhoB that is phosphorylated by activated PhoR
The translation is the whole process by which the nucleotide sequence of an mRNA is used to order and to join the amino acids in a polypeptide chain
Messenger RNA (mRNA)
Carries the genetic information transcribed from DNA in the form of a series of three-nucleotide sequences - called codons (each specifies a particular amino acid.
Transfer RNA (tRNA)
Very important to decipher the codons in mRNA. Each type of amino acid has its own subset of tRNAs, which bind the amino acid and carry it to the growing end of a polypeptide chain if the next codon in the mRNA calls for it.
Ribosomal RNA (rRNA)
Associates with a set of proteins to create ribosomes. These structures (physically) move along an mRNA molecule, catalyze the assembly of amino acids into polypeptide chains. They bind tRNAs and various other accessory proteins necessary for protein synthesis.
These types of RNA participate in translation in all cells
The development of three functionally distinct RNAs was probably the molecular key to the origin of life
The genetic code used by cells is a triplet code, with every three-nucleotide sequence, or codon, being “read” from a specified starting point in the mRNA
A total of 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop codons
The different codons for a given amino acid are said to be synonymous
Synthesis of all polypeptide chains in prokaryotic and eukaryotic cells begins with the amino acid methionine
In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionally is used as an initiator codon for methionine in eukaryotes
The three codons UAA, UGA, and UAG do not specify amino acids but constitute stop (termination) codons that mark the carboxyl terminus of polypeptide chains in almost all cells
The sequence of codons that runs from a specific start codon to a stop codon is called a reading frame
The linear array of ribonucleotides in groups of three in mRNA specifies the precise linear sequence of amino acids in a polypeptide chain
Particular mRNA theoretically could be translated in three different reading frames
The meaning of each codon is the same in most known organisms (a strong argument that life on earth evolved only once)
The genetic code has been found to differ for a few codons in many mitochondria, in ciliated protozoans, and in Acetabularia, a single-celled plant
Translation/decoding of the four nucleotide language of DNA and mRNA into the 20 amino acid language of proteins requires tRNA and enzymes called aminoacyl-tRNA synthetases
tRNA molecule must become chemically linked to a particular amino acid via a high-energy bond, forming an aminoacyl-tRNA; the anticodon in the tRNA then base-pairs with a codon in mRNA so that the activated amino acid can be added to the growing polypeptide chain to participate in protein synthesis
Some 30–40 different tRNAs have been identified in bacterial cells and as many as 50–100 in animal and plant cells
The function of tRNA molecules, which are 70–80 nucleotides long, depends on their precise three-dimensional structures
The four stems are short double helices stabilized by Watson-Crick base pairing; three of the four stems have loops containing seven or eight bases at their ends
While the remaining, unlooped stem contains the free 3 and 5 ends of the chain
The first and second bases of a codon almost always form standard Watson-Crick base pairs with the third and second bases, of the corresponding anticodon
But four nonstandard interactions can occur between bases in the wobble position
Adenine rarely is found in the anticodon wobble position, many tRNAs in plants and animals contain inosine (I), a deaminated product of adenine, at this position
Inosine can form nonstandard base pairs with A, C, and U
A tRNA with inosine in the wobble position thus can recognize the corresponding mRNA codons with A, C, or U in the third (wobble) position
The first step in decoding the genetic message: attachment of the appropriate amino acid to a tRNA, is catalyzed by a specific aminoacyl-tRNA synthetase
Second step: Recognition of the codon or codons specifying a given amino acid by a particular tRNA
Each of the 20 different synthetases recognizes one amino acid and all its compatible, or cognate, tRNAs
Some amino acids are so similar structurally, aminoacyl-tRNA synthetases sometimes make mistakes
Can be corrected but, by the enzymes themselves which have a proofreading activity that checks the fit in their amino acid binding pocket
Of the many components that participate in translating mRNA had to interact in free solution
The likelihood of simultaneous collisions occurring would be really low
Small proteins of 100–200 amino acids are therefore made in a minute or less
However, it takes 2–3 hours to make the largest known protein (titin) which is found in muscle and contains about 30,000 amino acid residues
The electron microscope helped ribosomes to first be discovered as small, discrete, RNA-rich particles in cells that secrete large amounts of protein
A ribosome is composed of three (in bacteria) or four (in eukaryotes) different rRNA molecules and as many as 83 proteins, organized into a large subunit and a small subunit
The small ribosomal subunit contains a single rRNA molecule, referred to as small rRNA
The large subunit contains a molecule of large rRNA and one molecule of 5S rRNA, plus an additional molecule of 5.8S rRNA invertebrates
The sequences of the small and large rRNAs from several thousand organisms are now known
Although the primary nucleotide sequences of these rRNAs vary considerably, the same parts of each type of rRNA theoretically can form base-paired stem-loops
Which would generate a similar three-dimensional structure for each rRNA in all organisms
During translation, a ribosome moves along an mRNA chain, interacting with various protein factors and tRNAs and very likely undergoing large conformational changes
The AUG codon for methionine functions as the start codon in the vast majority of mRNAs
A critical aspect of translation initiation is to begin protein synthesis at the start codon, thereby establishing the correct reading frame for the entire mRNA
Both prokaryotes and eukaryotes contain two different methionine tRNAs: tRNAiMet can initiate protein synthesis, and tRNAMet can incorporate methionine only into a growing protein chain
The first stage of translation: a ribosome assembles, complexed with an mRNA and an activated initiator tRNA
Which is correctly positioned at the start codon
A translation preinitiation complex is formed when the 40S subunit–eIF3 complex is bound by eIF1A and a ternary complex of the Met-tRNAiMet, eIF2, and GTP
Cells can regulate protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP
The phosphorylated complex is unable to exchange the bound GDP for GTP and cannot bind Met-tRNAiMet, inhibiting protein synthesis
The mRNA-eIF4 complex then associates with the preinitiation complex through an interaction of the eIF4G subunit and eIF3, forming the initiation complex
Scanning stops when the tRNAiMet anticodon recognizes the start codon
Which is the first AUG downstream from the 5’ end in most eukaryotic mRNAs
Recognition of the start codon leads to hydrolysis of the GTP associated with eIF2, an irreversible step that prevents further scanning
Once the small ribosomal subunit with its bound Met-tRNAiMet is correctly positioned at the start codon, union with the large (60S) ribosomal subunit completes the formation of an 80S ribosome
This requires the action of another factor (eIF5) and hydrolysis of a GTP associated with it
The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5 capped end as just described
But, some cellular mRNAs contain an internal ribosome entry site (IRES) located far downstream of the 5’ end
The correctly positioned eukaryotic 80S ribosome–Met-tRNAiMet complex is now ready to begin the task of stepwise addition of amino acids by the in-frame translation of the
mRNA
Special proteins called elongation factors (EFs) require to carry out the process of chain elongation
The key steps in elongation are the entry of each succeeding aminoacyl-tRNA, formation of a peptide bond, and the movement, or translocation, of the ribosome one codon at a time along the mRNA
The completion of translation initiation, as noted already, Met-tRNAiMet is bound to the P site on the assembled 80S ribosome
This region is called the P site - because the tRNA is chemically liked to the growing polypeptide chain
The second (aminoacyl-tRNA) is brought into the ribosome as a ternary
complex in association with EF1 ox- GTP and becomes bound to the A site, so named because it is where aminoacylated tRNAs bind
The hydrolysis of GTP promotes a conformational change in the ribosome that leads to tight binding of the aminoacyl-tRNA in the A site and release of the resulting EF1ox-GDP complex
With the initiating Met-tRNAiMet at the P site and the second aminoacyl-tRNA tightly bound at the A site
The amino group of the second amino acid reacts with the “activated” (ester-linked) methionine on the initiator tRNA, forming a peptide bond
Following peptide bond synthesis, the ribosome is translocated along the mRNA at a distance equal to one codon
This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2ox-GTP
As a result of translocation, tRNAiMet, now without its activated methionine, is moved to the E (exit) site on the ribosome; concurrently
The second tRNA, now covalently bound to a dipeptide (a peptidyl-tRNA), is moved to the P site
The locations of tRNAs bound at the A, P, and E sites are visible in the recently determined crystal structure of the bacterial ribosome
Base pairing is apparent between the tRNAs in the A and P sites with their respective codons in mRNA
The final stage of translation, like initiation and elongation, requires highly specific molecular signals that decide the fate of the mRNA–ribosome–tRNA-peptidyl complex
Two types of specific protein release factors (RFs) have been discovered
Eukaryotic eRF1, whose shape is similar to that of tRNAs, apparently acts by binding to the ribosomal A site and recognizing stop codons directly
The second eukaryotic release factor, eRF3, is a GTP-binding protein
After its release from the ribosome, a newly synthesized protein folds into its native three-dimensional conformation, a process facilitated by other proteins called chaperones
Hydrolysis of the bound GTP is thought to cause conformational changes in the GTPase itself or other associated proteins that are critical to various complex molecular processes
The translation of a single eukaryotic mRNA molecule to yield a typical-sized protein takes 30–60 seconds
Two phenomena significantly increase the overall rate at which cells can synthesize a protein:
The simultaneous translation of a single mRNA molecule by multiple ribosomes and rapid recycling of ribosomal subunits after they disengage from the 3’ end of an mRNA
Simultaneous translation of an mRNA by multiple ribosomes is readily observable in electron micrographs and by sedimentation analysis, revealing mRNA attached to multiple ribosomes bearing nascent growing polypeptide chains
Structures that are referred to as polyribosomes (or polysomes) were seen to be circular in electron micrographs of some tissues
Multiple copies of a cytosolic protein are found in all eukaryotic cells
Analogous to RNA, DNA is synthesized from deoxynucleoside 5’-triphosphate precursors (dNTPs)
DNA synthesis always proceeds in the 5’ → 3’ direction
Because chain growth results from the formation of a phosphodiester bond between the 3 oxygen of a growing strand and the phosphate of a dNTP
DNA polymerases can’t initiate chain synthesis de novo; instead, they require a short, preexisting RNA or DNA strand, called a primer, to begin chain growth
For DNA to function properly as a template during replication the two intertwined strands must be unwound or melted
To make the bases available for base pairing with the bases of the dNTPs that are polymerized into the newly synthesized daughter strands
The DNA region at which all these proteins come together to carry out the synthesis of daughter strands is called the replication fork, or growing fork
Synthesis of one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5’→3’ direction
The same direction as the movement of the replication fork (or growing fork)
There is a problem that comes in the synthesis of the other daughter strand
Called lagging strand
Eukaryotic proteins that participate in DNA replication has come largely from studies with small viral DNAs, particularly SV40 DNA, the circular genome of a small virus that infects monkeys
All other proteins involved in SV40 DNA replication are provided by the host cell
After parental DNA is separated into single-stranded templates at the replication fork, it is bound by multiple copies of RPA (replication protein A), a heterotrimeric protein
Both parental DNA strands that are exposed by local unwinding at a replication fork are copied into a daughter strand
The general consensus is that all prokaryotic and eukaryotic cells employ a bidirectional mechanism of DNA replication
Taking SV40 DNA for example, replication is initiated by binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins to form two replication forks
Activation of MCM helicase activity, which is required to initiate cellular DNA replication, is regulated by specific protein kinases called S-phase cyclin-dependent kinases
Viruses cannot reproduce by themselves and must commandeer a host cell’s machinery to synthesize viral proteins and in some cases to replicate the viral genome
RNA viruses, which usually replicate in the host-cell cytoplasm, have an RNA genome, and DNA viruses, which commonly replicate in the host-cell nucleus, have a DNA genome
Viral genomes may be single- or double-stranded, depending on the specific type of virus
The entire infectious virus particle, called a virion, consists of nucleic acid and an outer shell of protein
The surface of a virion contains many copies of one type of protein that binds specifically to multiple copies of a receptor protein on a host cell
A virus that infects only bacteria is called a bacteriophage, or simply a phage
The nucleic acid of a virion is enclosed within a protein coat, or capsid, composed of multiple copies of one protein or a few different proteins
Each of which is encoded by a single viral gene
A capsid plus the enclosed nucleic acid is called a nucleocapsid
Nature has found two basic ways of arranging the multiple capsid protein subunits and the viral genome into a nucleocapsid
In some viruses, multiple copies of a single coat protein form a helical structure that encloses and protects the viral RNA or DNA
Which runs in a helical groove within the protein tube
The number and arrangement of coat proteins in icosahedral, or quasi-spherical, viruses differ somewhat depending on their size
In large quasi-spherical viruses, each face of the icosahedron is composed of more than three subunits
In the smaller viruses (e.g.poliovirus), clefts that encircle each of the vertices of the icosahedral structure interact with receptors on the surface of host cells during infection
In the larger viruses (e.g., adenovirus), long fiber-like proteins extending from the nucleocapsid interact with cell-surface receptors on host cells
Many DNA bacteriophages, the viral DNA is located within an icosahedral “head” that is attached to a rodlike “tail”
During infection, viral proteins at the tip of the tail bind to host-cell receptors, and then the viral DNA passes down the tail into the cytoplasm of the host cell
The number of infectious viral particles in a sample can be quantified by a plaque assay
This assay is performed by culturing a dilute sample of viral particles on a plate covered with host cells and then counting the number of local lesions, called plaques, that develop
Since all the progeny virions in a plaque are derived from a single parental virus, they constitute a virus clone
This type of plaque assay is in standard use for bacterial and animal viruses
Plant viruses can be assayed similarly by counting local lesions on plant leaves inoculated with viruses
The lytic cycle of growth proceeds through the following general stages
1. Adsorption—Virion interacts with a host cell by binding multiple copies of capsid protein to specific receptors on the cell surface.
2. Penetration—Viral genome crosses the plasma membrane. For animal and plant viruses, viral proteins also enter the host cell.
3. Replication—Viral mRNAs are produced with the aid of the host-cell transcription machinery (DNA viruses) or by viral enzymes (RNA viruses). For both types of viruses, viral mRNAs are translated by the host cell translation machinery. Production of multiple copies of the viral genome is carried out either by viral proteins alone or with the help of host-cell proteins
4. Assembly—Viral proteins and replicated genomes associate to form progeny virions.
5. Release—Infected cells either ruptures suddenly (lysis), releasing all the newly formed virions at once, or disintegrates gradually, with a slow release of virions
Viral capsid proteins generally are made in large amounts because many copies of them are required for the assembly of each progeny virion
In each infected cell, about 100–200 T4 progeny virions are produced and released by lysis
In most such viruses, the DNA genome is transported (with some associated proteins) into the cell nucleus
Once inside the nucleus, the viral DNA is transcribed into RNA by the host’s transcription machinery
Most plant and animal viruses with an RNA genome do not require nuclear functions for lytic replication
In some of these viruses, a virus-encoded enzyme that enters the host during penetration transcribes the genomic RNA into mRNAs in the cell cytoplasm
mRNA is directly translated into viral proteins by the host cell translation machinery
One or more of these proteins then produces additional copies of the viral RNA genome
Lastly, progeny genomes are assembled with newly synthesized capsid proteins into progeny virions in the cytoplasm
After the synthesis of hundreds to thousands of new virions has been completed, most infected bacterial cells and some infected plant and animal cells are lysed, releasing all the virions at once
Some bacterial viruses, called temperate phages, can establish a nonlytic association with their host cells that do not kill the cell
The integrated viral DNA, called a prophage, is replicated as part of the cell’s DNA from one host-cell generation to the next
This phenomenon is referred to as lysogeny
The genomes of a number of animal viruses also can integrate into the host cell genome
The most important are the retroviruses, which are enveloped viruses with a genome consisting of two identical strands of RNA
In the retroviral life cycle, a viral enzyme called reverse transcriptase initially copies the viral RNA genome into single-stranded DNA complementary to the virion RNA; the same enzyme then catalyzes the synthesis of a complementary DNA strand
The resulting double-stranded DNA is integrated into the chromosomal DNA of the infected cell
The integrated DNA is called a provirus
Some retroviruses contain cancer-causing genes (oncogenes), and cells infected by such retroviruses are oncogenically transformed into tumour cells
