Assignment On Regulation of Transcription, Lytic Lysogeny Cascade and SOS Regulatory System

Assignment on Regulation of Transcription, Lytic Lysogeny cascade and SOS regulatory system
When isolated from bacteria, prokaryotic RNA polymerase has two forms: The coreenzyme and the holoenzyme. The core enzyme is a tetramer whose composition is given as 2 (two alpha subunits, one beta subunit, and one beta-prime subunit). Core RNA polymerase is capable of faithfully copying DNA into RNA but does not initiate at the correct site in a gene. That is, it does not recognize the promoter specifically. Correct promoter recognition is the function of the holoenzyme form of RNApolymerase.
Figure 1 The RNA polymerase holoenzyme contains another subunit, s( sigma), in addition to the subunits found in the core enzyme. Holoenzyme, 2, is capable of correct initiation at the promoter region of a gene. Sigma thus must be involved in promoter recognition. Sigma subunits are related but distinct in different forms of RNA polymerase holoenzyme. These specialized subunits direct RNA polymerase to promoter sequences for different classes of genes. For example, bacteria exposed to high temperatures synthesize a set of protective proteins called heat-shock proteins. The genes for the heat-shock proteins have special promoter sequences that are recognized by an RNA polymerase holoenzyme with a specific subunit. The discussed here is the major of the common bacterium E. coli, about which most is known. Promoter recognition RNA polymerase holoenzyme starts by recognizing the promoter of a gene. The promoter isn't copied into RNA, but it is, nonetheless, an important piece
Submitted by: Shraddha Bhatt. Page 1
of genetic information. The information in a promoter was determined by lining up a large number of promoters and counting how many times a particular base appeared at a given position in the various promoter sequences. The consensus sequence is given by the statistically most probable base at each pointthe bases that appear most often in the promoter collection. Very few, if any, naturally occurring promoters match the consensus sequence exactly, but the strength of a promoter (how actively RNA polymerase initiates at it) correlates well with the degree of consensus match. For example, the promoters of genes for ribosomal RNA match the consensus well, while the promoters for the mRNA encoding some regulatory proteins match the consensus poorly. This correlates with the relative amounts of each gene product that are needed at any one time: many ribosomes, and only a few regulatory proteins. The consensus sequence for an E. coli promoter has two conserved regions near positions -35 and -10 relative to the transcription start site. That is, the template-directed synthesis of RNA begins 35 base pairs downstream of the first consensus region and ten base pairs downstream of the second. The -35 consensus is: TTG ACA. The -10 consensus is: TATAA T. A couple of important points exist about the consensus. First, not all bases in the consensus are conserved to the same amount. The bases marked with bold type and underlined are more conserved than the others, and the -10 region is more conserved overall than is the -35 region. Secondly, the promoter sequence is asymmetrical; that is, it reads differently in one direction than in the other. (Compare this to the recognition sequence for the restriction enzyme BamHI, GGATCC.) This asymmetry means that RNA polymerase gets directional information from the promoter in addition to information about the starting point for transcription. The transcription process RNA polymerase only goes one direction from a promoter and only one strand of DNA is used as a template at any one time. To provide this template strand, the initiation of transcription involves a short unwinding of the DNA double helix. This is accomplished in a two-step fashion. First, RNA polymerase binds to the promoter to form the closed complex, which is relatively weak. Then, the double-stranded DNA goes through a conformational change to form the much stronger open complex through opening of the base pairs at the -10 sequence, as shown in Figure 2 .
Submitted by: Shraddha Bhatt.
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Figure 2 The initiator nucleotide binds to the complex and the first phosphodiester bonds are made, accompanied by release of . The remaining core polymerase is now in the elongation mode. Several experimental observations support the picture presented in the next figure, namely the fact that less than one exists in the cell per core enzyme in each cell.
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Figure 3 Elongation is the function of the RNA polymerase core enzyme. RNA polymerase moves along the template, locally unzipping the DNA double helix. This allows a transient base pairing between the incoming nucleotide and newly-synthesized RNA and the DNA template strand. As it is made, the RNA transcript forms secondary structure through intra-strand base pairing. The average speed of transcription is about 40 nucleotides per second, much slower than DNA polymerase. Other protein factors may bind to polymerase and alter the rate of transcription and some specific sequences are transcribed more slowly than others are. Eventually, RNA polymerase must come to the end of the region to be transcribed. Termination of transcription in vitro is classified as to its dependence on the protein factor, rho (). Rho-independent terminators have a characteristic structure, which features (a) A strong G-C rich stem and loop, (b) a sequence of 46 U residues in the RNA, which are transcribed from a corresponding stretch of As in the template. Rho-factor-dependent terminators are less well defined, as shown in Figure 4 .
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Overall, the process of RNA synthesis in eukaryotes is similar to that of prokaryotes. There are some real differences, however. For one thing, initial transcripts in eukaryotes contain introns, which must be removed after transcription (this will be examined later). Eukaryotes also have three RNA polymerases, instead of just one. Each of these polymerases transcribes a different class of genes, as outlined in the table below: RNA Polymerase Genes encoding ribosomal I RNA RNA polymerase Genes encoding II messenger RNA RNA Polymerase Genes encoding transfer III RNA Eukaryotic transcription will focus on genes transcribed by RNA polymerase II (known as class II genes). As with prokaryotes, the transcription process can be broken down into the steps of initiation, elongation, and termination. In eukaryotes, there is also the additional step of RNA processing, which occurs during and after transcription.
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Initiation Initiation in eukaryotes is much more complex than it is in prokaryotes. Eukaryotic genes must be much more carefully regulated, because many genes are only expressed in specific cells or tissues at specific times in the organism's life. To achieve this careful regulation, eukaryotes have evolved a more complicated initiation scheme than prokaryotes. In addition to promoters, eukaryotic genes also have regulatory regions called enhancers. Both elements (promoter and enhancer) are required for full, correct expression of eukaryotic genes. As a result of this added complexity, eukaryotic RNA polymerases do not have anything equivalent to the sigma subunit found in prokaryotic RNA polymerases. Instead, eukaryotes have groups of transcription factors, which are proteins, independent of the RNA polymerases, that recognize promoter and enhancer sequences. Eukaryotic promoters, like prokaryotic promoters, contain conserved sequences that are important for initiation. (Eukaryotes, because of their added complexity, tend to have more conserved sequences in their promoters than do prokaryotes.) One important sequence in most eukaryotic promoters is found around -30, and has the sequence TATAAA (or something close to it). This promoter element, known as the TATA Box, is analogous to the -10 element in prokaryotes. Other promoter sequences vary from gene to gene, but a common one is GGCCAATCT, otherwise known as the CCAAT Box (for the central bases in the sequence), which tends to occur around -80. A group of basal transcription factors helps to initiate transcription of class II genes. Each member of this group is named "TFII" for Transcription Factor, class II genes. The individual factors are assigned a separate letter designation. For example, TFIID, a factor made of multiple polypeptides, recognizes and binds to the TATA box. This factor and the other factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, and TFIIJ) forms a complex on the DNA that recruits RNA polymerase II to the promoter, and promotes initiation of transcription. These transcription factors are sufficient to get a basal (minimal) level of transcription. Other transcription factors binding to other promoter and enhancer elements are necessary for higher levels of transcription. Elongation Elongation in eukaryotes is just like in prokaryotes. Termination Termination in eukaryotes is quite different in eukaryotes than it is in
prokaryotes. Eukaryotic genes have no strong termination sequences like prokaryotes. Instead, RNA polymerase II continues transcribing up to 1000 to 2000 nucleotides beyond where the 3' end of the mature mRNA will be. The actual 3' end will be determined during RNA processing. Processing Eukaryotic class II transcripts are processed in order to produce the final mRNA. Processing of the initial transcript includes capping, polyadenylation, and intron removal. Ribosomal and transfer RNAs are also processed, but differently; they are neither capped nor polyadenylated. Capping of the RNA occurs at the 5' end. A methylated guanine nucleotide is added to the transcript in a 5' to 5' phosphodiester linkage (it's like a nucleotide added to the 5' end in the backwards direction). This 'cap' is important for recognition of the mRNA by ribosomes during translation. Polyadenylation involves cleavage of the RNA to produce the proper 3' end, and addition of a string of adenine nucleotides. The position of the 3' end is determined by a sequence within the RNA itself. This sequence, AAUAAA, is known as the polyadenylation signal. When this signal is recognized by the appropriate enzymes, the RNA is cleaved 10 to 30 nucleotides downstream of the signal, and a series of adenine nucleotides is added. This polyadenylation is done without a template - the As are simply added one after another to the 3' end of the RNA. This poly (A) tail, which averages about 200 nucleotides in length, helps protect the RNA from degradation, and plays other regulatory roles that are beyond the scope of our discussion. Introns in some RNAs (particularly mitochondrial RNAs) are capable of self-splicing (or autocatalytic splicing). In these splicing reactions, no protein enzyme is required - the enzyme activity resides within the intron RNA itself! Such RNA enzymes are termed ribozymes. Class II RNAs (pre-mRNAs) from most eukaryotes, however, do require protein enzymes to remove their introns. The splicing of these RNAs is carried out by large protein/RNA complex called spliceosomes. Spliceosomes are made up of five different snRNPs (pronounced 'snurps'; short for small nuclear ribonucleoprotein), called U1, U2, U4, U5, and U6. Each snRNP consists of a specific small nuclear RNA (snRNA) molecule complexed with protein. Spliceosomes are able to detect intron/exon boundaries, cleave the RNA at the appropriate point, and join adjacent exons together to produce the mature mRNA. Transcription: Summary of Key Points
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Transcription is carried out by enzymes called RNA polymerases, which synthesize RNA in a 5' to 3' direction. Prokaryotes have one RNA polymerase; eukaryotes have three RNA polymerases, each of which transcribes a different class of gene. Transcription occurs in three basic steps: initiation, elongation, and termination. Initiation in prokaryotes involves the recognition of promoter sequences by the sigma subunit of RNA polymerase. Initiation in eukaryotes involves the recognition of promoter sequences by transcription factors, which then recruit RNA polymerase to the promoter. Termination in prokaryotes occurs at termination sequences. Depending upon the gene, termination may or may not involve the termination protein rho. Termination in eukaryotes is less specific: it occurs well downstream of the 3' end of the mature mRNA. Eukaryotic transcripts must be processed to produce mature mRNAs; Prokaryotic RNAs are not processed. Processing involves the addition of a 5' cap, the production of the correct 3' end by cleavage and polyadenylation, and the removal of introns by spliceosomes.
Lytic Cascade in Lambda ()Phage Lambda () bacteriophage exhibits one of the most interesting, but intricate cascade circuits for the twoalternative pathways (lytic
cycle or lysogeny), that it can follow after infection. A brief mention of this phage and its mode of infection was earlier made in Sexuality and Recombination in Bacteria and Viruses, where it was also explained that the temperate phages like lambda can be classified as episomes, since these are dispensible and exist in free as well as integrated states. The phage can infect bacteria and multiply in the normal manner leading to lysis, but this mode can shift to lysogeny where the phage DNA gets integrated with host DNA and stays tnere 'as prophage till again it becomes lytic. "How are these two states interconnected, and how does one state shift to another" are the subject of regulation of gene expression. A map of lambda ()DNA is given in Figure 1, which has early genes in the middle and late genes at both ends of linear DNA. The linear DNA takes a circular shape duringinfection, so that the late genes from both ends come together in a cluster to form one transcription unit. When the phage infects a host cell, the early and delayed early genes of the phage are expressed irrespective of whether the phage has to follow lytic or lysogenic mode of development. At this stage, a decision about alternative pathways of development is taken. If the late genes are expressed at this stage, lytic cycle sets in, but if^a regulator gene (cI) synthesizing a repressor is expressed, the late genes can not be expressed and lysogeny sets in (Fig. 2). It is interesting to note that repressor protein coded by cI regulates its own synthesis also by working as an activator, so that if repressor is inactivated, its further synthesis does not take place and the phage is forced to enter a lytic cycle.Mutants in the gene cI cannot maintain lysogeny, since then the late' genes can not be stopped from being expressed, due to the absence of active repressor.
Fig:1
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Fig:2
Three sets of genes in lambda ()phage Immediate early cro and N genes and their control region (PR/OR and PL/OL). As soon as the phage infects, transcription is initiated with the help of two promoters, PR on the right side and PL on the left side. The transcription starts on both DNA strands, one strand being transcribed on the left side and the other on the right side. There are two immediate early genes cro and N, cro being transcribed on the right side under the control of PR and N being transcribed on the left side under the control of PL.When transcribed, cro has dual function, namely (i) it prevents synthesis of repressor by cI (an action which is necessary for lytic cycle), which otherwise causes lysogeny and (ii) it turns off the expression of the immediate early genes including its ownself, since their expression is not needed later in the lytic cycle. Gene N, as earlier discussed in Expression of Gene : Protein Synthesis 2. Transcription in Prokaryotes and Eukaryotes, codes for an antiterminator pN (pN = product of gene N), which allows transcription to proceed into the delayed early genes without the need of any fresh promoter or operator genes. Associated with promoters PR and PLare operators OR and OL respectively which are sites for repressor binding to prevent RNA polymerasefrom initiating transcription (Fig. 1). The gene cI(regulator gene coding for repressor) lies in betweenPR/OR and PL/OL control regions and when active, it not only inhibits the expression of cro and Nbut also promotes its own expression (Fig. 3), so that the repressor has a dual function and also works as an activator for its
own autogenous expression and regulation.
Fig:3
Delayed early genes (cIIcIII and Q)and their control on lysogeny and lytic cycle. Among the delayed early genes, regulators cII (on the right) and cIII (on the left) regulate synthesis of repressor by cl, to allow phage enter lysogeny (Figs. 1,2). On the other hand, a gene Q (on the right) is a regulator which acts as anantiterminator, and allows the transcription of late genes meant for lysis. The two pathways (lytic and lysogenic) are so intimately related that it is difficult to predict which pathway will be followed and how is the decision for two alternative pathways is actually taken. This will be further discussed later in this section. Late genes for lysis (right end) and, tail and head genes (left end). In the phage particle, the late genes are found at the two ends of the linear DNA molecule, genes 5 and R on the right end, and the tail and head genes on the left end. Genes for recombination are also found on the left side of cIII and are transcribed as late genes. Soon after the infection, the two ends of DNA molecule join to form a ring, so that all late genes are arranged in a single group containing S-R genes from right end and head and tail genes A to J from left end (Fig. 1). There is a promoter gene PR between genes Q and S. In the absence of pQ (product of Q gene), the transcription from PRis constitutive, but terminates at tR3 lying close to PR giving a product, 6S RNA (194 bases
long). However, when pQ is present, it suppresses tR3 and 6S RNA is extended with the result that late genes are heavily expressed. Thus, Q gene induces transcription of late genes, which continue to be transcribed all through their length. On the left side also, late genes continue to be transcribed. Transcription from both sides stops on the ring molecule, before the RNA polymerasemolecules from two sides could clash. Anti-repressor (product of cro gene) for lytic cycle We have earlier discussed that cro gene has dual function of preventing the synthesis of repressor and of turning off the early genes. It codes for a protein (9000 daltons) which forms a dimer and acts on promoter PM (for cI gene), PL (for N gene on the left side) and PR (for cro gene) itself, so that it stops expression of repressor gene cI as well as that of the early genes (Figs. 2,4) including N on the left and cro on the right. This fulfils the early requirement for establishment of lytic cycle, because if cI is expressed, this will stop expression of late genes and thus will establish lysogeny.
Fig:4
Lysogeny through establishment and maintenance of repressor (promoters PE and PM) In phage lambda (), a very delicate balance is maintained between lytic cycle and lysogeny, with a very sophisticated and intricate circuit of events, which will be briefly described in this section. It will be seen that the repressor's synthesis is first established with the help of a promoter PE under the control
of genes cII and cIII,and is subsequently maintained by the repressor itself through a promoter PM. Promoter PE (or PRE)for repressor establishment and the clear plaque genes (cI, cII, cIII). There is apromoter gene PE lying between cro and cII genes. The product of genes cII and cIII act on the promoter PE, so that the RNA polymerase ' enzyme can initiate transcription towards the left thus synthesizing RNA on genes croand cI. This transcript will consist of (i) the transcript from the antisense strand of cro gene, which can not be translated (cro gene is transcribed in the right direction from PR to yield translatable mRNA) and (ii) mRNA from clgene which can be translated to give rise to repressor (Fig. 5). The efficiency of synthesis of repressor from cIthrough PE is 7-8 times the synthesis from the promoter PM (see next section for further details). This repressor synthesized under the control of PE, binds immediately to the operators OL and OR thus inhibiting transcription from PL and PR, leading to turning off the expression of all phage genes. This also halts the synthesis of cII and cIII proteins. Since cII and cIII proteins are unstable and rapidly decay, PE can not be used any longer for synthesis of repressor. The repressor is now synthesized under the influence of PM due to the positive control of repressor (already synthesized under the control of promoter PE), which binds on OR (which also contains the promoter PR).
Fig:5
The genes cI, cII and cIII are so named, because mutants in these genes produce clear plaques, due to the presence of only lysed cells. In the wild type phage, when the lysis occurs, due to the presence of some lysogenic bacteria, clear plaques are not obtained (plaque is a region on the culture medium on the plate, generated due to lysis of bacterial cells). Mutants in cI (cI-)differ from the mutants cII-and cIII-, since the former can neither establish, nor maintain lysogeny. The mutants cII- and cIII- have difficulty in
establishing lysogeny, but once established, lysogeny can be maintained. This suggested that the products of genes cII and cIIIare needed only for the establishment of lysogeny, but not for its maintenance. The genes cII and cIIIare thus needed to circumvent the difficulty of autogenous circuit of cI, which can not start the synthesis of repressor de novo. The different steps involved in establishment and autogenous maintenance of lysogeny are shown in Figure 6.
Fig:6
Promoter PM (PRM)for autogenous maintenance of repressor. Once the synthesis of repressor is established through the action of cII and cIII gene products on PE , these genes (cII and cIII)are switched off by the action of repressor on OR and OL.But the repressor works as an activator by binding on OR, which also contains the promoter PM (also known PRM since it is on the right side of cI). By acting on OR, the repressor helps RNA polymerase to bind on PM and start further synthesis of repressor. In a remarkable manner it also controls the concentration of repressor, so that when repressor concentration is too high, its further synthesis can be stopped.
Autoregulation of repressor synthesis by cooperative binding of repressor. Each operator (OR and OL)has three binding sites, OR with OR1-OR2OR3and OL with OLl-OL2-OL3.In each case, site 1 is closest to its promoter (PR and PL)and site 3 is farthest. Site 1 has greater affinity (roughly tenfold) for repressor than other sites in each operator, so that the repressor first binds to OR1and OL1. The repressor functions as dimers for maintenance of lysogeny, and at low concentration it binds only to sites OR1and OL1, while with increasing concentration, the repressor dimers can also bind to sites 2(OR2 and OL2)and 3(OR3 and OL3). Presence of repressor on site 1 increases its affinity for site 2 but this interaction does not proceed to site 3. When lysogeny is established, only sites 1 and 2 are occupied but site 3 is not occupied. Since PR lies within OR1and PL lies within OL1,occupancy of OR1OR2 and OL1-OL2 physically block PR and PLrespectively and inhibit the expression of all genes thus establishing lysogeny. The relationship of OR and PM is such that PM (site for RNA polymerase binding) lies rather close to OR2. When dimers are bound to OL1-O,2, the dimer at OR2 interacts with RNA polymerase in such a way that the latter can utilize PM for transcription (Fig. 7). It is interesting that a repressor dimer bound on OR2 can inhibit transcription from PR, but promote transcription from PM.
Fig:7
However, when repressor concentration increases to such an extent, that it may now occupy even IR3 in addition to OR1-OR2, the transcription from cI is prevented. This effect depends on the fact that PM lies within OR3and when OR3 is occupied by repressor, it is not available for RNA polymerase. When repressor synthesis stops due to occupancy of OR3 by repressor, the concentration of repressor consequently falls, and OR3 again becomes
available for RNA polymerase and synthesis of repressor starts once again due to the presence of repressor onOR2. Thus repressor is an autogenous regulator of its own synthesis causing the inhibition by negative control at its high concentration and promoting by positive control at its low concentration. Change from lysogeny to lytic cycle by cleavage of repressor dimers in the connector region. The repressor functions as a dimer and each unit (27,000 daltons) has (i) a N-terminal domain (1-92 residues), which binds to operator; (ii) a C-terminal end (133-132 residues), which enhances the affinity of N-terminal domain for binding and (iii) a connector region (93-132 residues). The induction of a lysogenic prophage to enter the lytic cycle is caused by cleavage of repressor in the connector region between 111 and 112 amino acid residues. The cleavage releases the C-terminal domains, so that Nterminal domains now do not have sufficient affinity to remain attached to the operator. N-terminal domains thus dissociate, leading to lytic infection, because now the genes repressed earlier due to repressor, will be expressed causing lysis (Fig. 8). This transition from lysogeny to lysis, involves the expression of Q gene with the help of promoter PR' giving its product pQ regulator which helps in the expression of late genes S and R (Fig. 4).
Fig:8
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Assignment on Regulation of Transcription, Lytic Lysogeny cascade and SOS regulatory system

Submitted by: Shraddha Bhatt.

Submitted by: Shraddha Bhatt.

Submitted by: Shraddha Bhatt.

Submitted by: Shraddha Bhatt.

Submitted by: Shraddha Bhatt.

Submitted by: Shraddha Bhatt.

Submitted by: Shraddha Bhatt.

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