Chapter 8

Major Shifts in Prokaryotic Transcription

8.1 8.1 Modification of The Host RNA Modification of The Host RNA Polymerase During Phage Polymerase During Phage

InfectionInfection SPO1(SPO1(B. subtilisB. subtilis phage, large DNA genome) phage, large DNA genome)

Temporal program of transcriptionTemporal program of transcription

Time of Time of infection infection

Genes Genes expressedexpressed

RNA polymeraseRNA polymerase

0 - 5′0 - 5′ Early genesEarly genes Host holoenzymeHost holoenzyme5 - 10 ′5 - 10 ′ Middle genesMiddle genes gp28+host coregp28+host core

10 - 10 - endend Late genesLate genes gp33+gp34+host gp33+gp34+host corecore

Figure 8.1 Temporal control of Figure 8.1 Temporal control of transcription In phage SPO1- transcription In phage SPO1-

infected B. subtilis.infected B. subtilis. (a)(a) Early transcription is directed by Early transcription is directed by the host RNA polymerase holoenzyme, the host RNA polymerase holoenzyme, including the host including the host σσ factor (blue); one of factor (blue); one of the early phage proteins is gp28 the early phage proteins is gp28 (green), a new (green), a new σσ factor. factor. (b)(b) Middle transcription is directed by Middle transcription is directed by gp28, in conjunction with the host core gp28, in conjunction with the host core polymerase (red); two middle phage polymerase (red); two middle phage proteins are gp33 and gp34 (purple and proteins are gp33 and gp34 (purple and yellow, respectively); together, these yellow, respectively); together, these constitute yet another constitute yet another σσ factor. factor. (c)(c) Late transcription depends on the Late transcription depends on the host core polymerase plus gp33 and 34.host core polymerase plus gp33 and 34.

Evidence for σ switching modelEvidence for σ switching model

Genetic studiesGenetic studies mutations in gene 28 prevent early-to-mutations in gene 28 prevent early-to-

middle switch; middle switch; mutations in gene 33 or 34 prevent mutations in gene 33 or 34 prevent

middle-to-late middle-to-late switchswitch Biochemical studiesBiochemical studies purification of RNA polymerasepurification of RNA polymerase

Figure 8.2 Subunit compositions of RNA polymerases in SP01 phage-Figure 8.2 Subunit compositions of RNA polymerases in SP01 phage-infected infected B. subtilisB. subtilis cells. cells.

Polymerases were separated by chromatography and subjected to SDS-Polymerases were separated by chromatography and subjected to SDS-PAGE to display their subunits. Enzyme B (first lane) contains the core PAGE to display their subunits. Enzyme B (first lane) contains the core subunits (subunits (ββ', ', ββ, and , and αα), as well as subunit IV (gp28). Enzyme C (second lane) ), as well as subunit IV (gp28). Enzyme C (second lane) contains the core subunits plus subunits V (gp33) and Vl (gp34). The last two contains the core subunits plus subunits V (gp33) and Vl (gp34). The last two lanes contain separated lanes contain separated δδ and and σσ subunits, respectively. subunits, respectively.

Figure 8.3 Specificities of polymerases B and C. Pero et al. measured polymerase specificity by transcribing SP01 DNA in vitro with core polymerase (a), enzyme B (b), or enzyme C (c), in the presence of [3H]UTP to label the RNA product. Next they hybridized the labeled RNA to SP01 DNA in the presence of each of the following competitors: early SP01 RNA (green) made in vivo in the presence of chloramphenicol (CAM); middle RNA (blue) collected from phage-infected cells at 10 minutes post-infection; and late RNA (red) collected from phage-infected cells 30 minutes post- infection, The product of the core polymerase is competed roughly equally by all three classes of RNA. On the other hand, competition for the product made by B plus δ is clearly competed best by middle RNA, and the product made by C plus δ is competed best by late RNA. These differences are not as dramatic as one might prefer, but they are easiest to see at low competitor concentration.

SUMMARY SUMMARY Transcription of phage SPO1 genes in infected Transcription of phage SPO1 genes in infected B. subtilisB. subtilis cells proceeds according to a temporal program in which cells proceeds according to a temporal program in which early genes are transcribed first, then middle genes, and early genes are transcribed first, then middle genes, and finally late genes. This switching is directed by a set of finally late genes. This switching is directed by a set of phage-encoded phage-encoded σσ factors that associate with the host core factors that associate with the host core RNA polymerase and change its specificity from early to RNA polymerase and change its specificity from early to middle to late. The host middle to late. The host σσ is specific for the phage early is specific for the phage early genes; the phage gp28 protein switches the specificity to genes; the phage gp28 protein switches the specificity to the middle genes; and the phage gp33 and gp34 proteins the middle genes; and the phage gp33 and gp34 proteins switch to late specificity.switch to late specificity.

8.2 8.2 The RNA Polymerase The RNA Polymerase Encoded in Phage T7Encoded in Phage T7

T7 T7 ((E. coliE. coli phage, small genome) phage, small genome)

Temporal control of transcription in T7Temporal control of transcription in T7genesgenes ExpressiExpressi

on stageon stageproductproduct

Class IClass I earlyearly Phage RNA Phage RNA polymerase, ect.polymerase, ect.

Class IIClass II middlemiddle Class II proteinsClass II proteinsClass IIIClass III latelate Class III proteinsClass III proteins

Figure 8.4 Temporal Figure 8.4 Temporal control of transcription in control of transcription in phage T7-infected E. coil.phage T7-infected E. coil.

(a) Early (class I) transcription (a) Early (class I) transcription depends on the host RNA depends on the host RNA polymerase holoenzyme, polymerase holoenzyme, including the host including the host σσ factor factor (blue); one of the early phage (blue); one of the early phage proteins is the T7 RNA proteins is the T7 RNA polymerase (green).polymerase (green). (b) Late (class II and III) (b) Late (class II and III) transcription depends on the T7 transcription depends on the T7 RNA polymerase.RNA polymerase.

SUMMARYSUMMARY Phage T7, instead of coding for a new Phage T7, instead of coding for a new σσ factor to factor to change the host polymerase's specificity from change the host polymerase's specificity from early to late, encodes a new RNA polymerase with early to late, encodes a new RNA polymerase with absolute specificity for the later phage genes. This absolute specificity for the later phage genes. This polymerase, composed of a single polypeptide, is a polymerase, composed of a single polypeptide, is a product of one of the earliest phage genes, gene 1. product of one of the earliest phage genes, gene 1. The temporal program in the infection by this The temporal program in the infection by this phage is simple. The host polymerase transcribes phage is simple. The host polymerase transcribes the earliest (class I) genes, one of whose products the earliest (class I) genes, one of whose products is the phage polymerase, which then transcribes is the phage polymerase, which then transcribes the later (class II and class III) genes.the later (class II and class III) genes.

8.3 8.3 Control of transcription Control of transcription During SporulationDuring Sporulation

Figure 8.5 Two types of B.subtilis cells. (a) B.subtilis vegatative cells and (b) a sporulating cell. With an endospore developing at the left end.

Figure 8.6 Map of part of plasmid p213. This DNA region contains two promoters: a vegetative promoter (Veg) and a sporulation promoter (0.4 kb). The former is located on a 3050 bp EcoRI-HincII fragment (blue); the latter is on a 770 bp fragment (red).

Figure 8.7 Specificities of σA and 6E. Losick and colleagues transcribed plasmid p213 in vitro with RNA polymerase containing σA (lane 1) or σE (lane 2). Next they hybridized the labeled transcripts to Southern blots containing EcoRI-Hincll fragments of the plasmid. As shown in Figure 8.6, this plasmid has a vegetative promoter in a 3050 bp EcoRI-Hincll fragment, and a sporulation promoter in a 770 bp fragment. Thus, transcripts of the vegetative gene hybridized to the 3050 bp fragment, while transcripts of the sporulation gene hybridized to the 770 bp fragment. The autoradiogram in the figure shows that the σA enzyme transcribed only the vegetative gene, while the σE enzyme transcribed both the vegetative and sporulation genes.

Figure 8.8 Specificity of Figure 8.8 Specificity of σσEE determined by determined by run-off transcription from the sporun-off transcription from the spollDllD

promoter.promoter. Rong et al. prepared a restriction fragment Rong et al. prepared a restriction fragment containing the spocontaining the spollDllD promoter and transcribed promoter and transcribed it in vitro with B. subtilis core RNA polymerase it in vitro with B. subtilis core RNA polymerase plus plus σσEE (middle lane) or (middle lane) or σσBB plus plus σσcc (right lane) (right lane) Lane M contained marker DNA fragments Lane M contained marker DNA fragments whose sizes are indicated at left The arrow at whose sizes are indicated at left The arrow at the right indicates the position of the expected the right indicates the position of the expected run-off transcript from the sporun-off transcript from the spollDllD promoter promoter (about 700 nt). Only the enzyme containing (about 700 nt). Only the enzyme containing σσEE made this transcript.made this transcript.

SUMMARY SUMMARY When the bacterium B. subtilis sporulates, a whole new set When the bacterium B. subtilis sporulates, a whole new set of sporulation-specific genes is turned on, and many, but not of sporulation-specific genes is turned on, and many, but not all, vegetative genes are turned off. This switch takes place all, vegetative genes are turned off. This switch takes place largely at the transcription level. It is accomplished by largely at the transcription level. It is accomplished by several new several new σσ factors that displace the vegetative factors that displace the vegetative σσ factor factor from the core RNA polymerase and direct transcription of from the core RNA polymerase and direct transcription of sporulation genes instead of vegetative genes. Each sporulation genes instead of vegetative genes. Each σσ factor factor has its own preferred promoter sequence.has its own preferred promoter sequence.

8.4 8.4 Genes with Multiple Genes with Multiple PromoterPromoter

The The B. subtilis spoVGB. subtilis spoVG Gene Gene The Anabaena Glutamine The Anabaena Glutamine

Synthetase GeneSynthetase Gene The The E. coli glnAE. coli glnA Gene Gene

Figure 8.10 Resolution of Figure 8.10 Resolution of RNA polymerases that RNA polymerases that

transcribe the spotranscribe the spoVGVG gene gene from two different from two different

promoters.promoters.

Figure 8.10 Resolution of RNA polymerases that transcribe the Figure 8.10 Resolution of RNA polymerases that transcribe the spospoVGVG gene from two different promoters. gene from two different promoters.

Losick and his colleagues purified polymerase from B. subtilis ceils that Losick and his colleagues purified polymerase from B. subtilis ceils that were running out of nutrients. The last purification step was DNA-cellutose were running out of nutrients. The last purification step was DNA-cellutose column chromatography. The polymerase activity in each fraction from the column chromatography. The polymerase activity in each fraction from the column is given by the red line and the scale on the left-hand y axis. The salt column is given by the red line and the scale on the left-hand y axis. The salt concentration used to remove the enzyme from the column is given by the concentration used to remove the enzyme from the column is given by the green line and the scale on the right-hand y-axis. The inset shows the results green line and the scale on the right-hand y-axis. The inset shows the results of a run-off transcription assay using a DNA fragment with two spoof a run-off transcription assay using a DNA fragment with two spoVGVG promoters spaced 10 bp apart, The fraction numbers at the top of the inset promoters spaced 10 bp apart, The fraction numbers at the top of the inset correspond to the fraction numbers from the column at bottom. The last lane correspond to the fraction numbers from the column at bottom. The last lane (M) contained marker DNA fragments. The two arrowheads at the left of the (M) contained marker DNA fragments. The two arrowheads at the left of the inset indicate the two run-off transcripts, approximately 110 and 120 nt in inset indicate the two run-off transcripts, approximately 110 and 120 nt in length. The column separated a polymerase that transcribed selectively from length. The column separated a polymerase that transcribed selectively from the downstream promoter and produced the shorter run-off transcript the downstream promoter and produced the shorter run-off transcript (fractions 19 and 20) from a polymerase that transcribed selectively from the (fractions 19 and 20) from a polymerase that transcribed selectively from the upstream promoter and produced the longer run-off transcript (fractions 22 upstream promoter and produced the longer run-off transcript (fractions 22 and 23).and 23).

Figure 8.11 Specificities of Figure 8.11 Specificities of σσ BB and and σσ EE. .

LOSiCk and colleagues LOSiCk and colleagues purified sigma factors purified sigma factors σ σ BB and and σσ EE by gel by gel electrophoresis and testedelectrophoresis and testedthem with core polymerase them with core polymerase by the same run-off by the same run-off transcription assay used in transcription assay used in Figure 8.10. Lane 2, Figure 8.10. Lane 2, containing containing σσ EE, caused , caused initiation selectively at the initiation selectively at the downstream promoter (P2). downstream promoter (P2). Lane 5, containing Lane 5, containing σσ BB, , caused initiation selectively caused initiation selectively at the upstream promoter at the upstream promoter (P1). Lane 6, containing both (P1). Lane 6, containing both σσ factors caused initiation at factors caused initiation at both promoters. The other both promoters. The other lanes were the results of lanes were the results of experiments with other experiments with other fractions containing neither fractions containing neither σσ factor.factor.

Figure 8.11 Overlapping promoters in B.subtills spoVG.Figure 8.11 Overlapping promoters in B.subtills spoVG. P1 denotes the upstream promoter, recognized by P1 denotes the upstream promoter, recognized by σ σ BB; the start of ; the start of transcription and -10 and -35 boxes for this promoter are indicated in red transcription and -10 and -35 boxes for this promoter are indicated in red above the sequence. P2 denotes the downstream promoter, recognized by above the sequence. P2 denotes the downstream promoter, recognized by σσ EE; the start of transcription and -10 and -35 boxes for this promoter are ; the start of transcription and -10 and -35 boxes for this promoter are indicated in blue below the sequence.indicated in blue below the sequence.

Summary Summary Some prokaryotic genes must be transcribed Some prokaryotic genes must be transcribed under conditions where two different under conditions where two different σσ factors factors are active. These genes are equipped with two are active. These genes are equipped with two different promoters, each recognized by one of different promoters, each recognized by one of the two the two σσ factors. This ensures their expression factors. This ensures their expression no matter which factor is present and allows for no matter which factor is present and allows for differential control under different conditions.differential control under different conditions.

8.5 8.5 The The E. coliE. coli Heat Shock Heat Shock GenesGenes

htpRhtpR gene, σ gene, σ32 32 (σ(σHH) ) Comparison of σComparison of σ32 and32 and σ σ70 70 gene:gene: -35 sequence space -10 -35 sequence space -10

sequencesequenceσσ7070 TTGACA 16-18 TATAA TTGACA 16-18 TATAAσσ3232 CNTTGAA 13-15 CCCCATNT CNTTGAA 13-15 CCCCATNT

SUMMARYSUMMARY The heat shock response in E. coli is The heat shock response in E. coli is governed by an alternative governed by an alternative σσ factor, factor, σσ 3232 ( (σσ HH) ) which displaces which displaces σσ 7070 ( (σσ AA) and directs the RNA ) and directs the RNA polymerase to the heat shock gene promoters.polymerase to the heat shock gene promoters. The accumulation of The accumulation of σσ 3232 in response to in response to high temperature is due to stabilization of high temperature is due to stabilization of σσ 3232 and enhanced translation of the mRNA and enhanced translation of the mRNA encoding encoding σσ 3232 . .

8.6 Infection of 8.6 Infection of E. coliE. coli by by Phage λPhage λ

Phage lambda can replicate in either of two ways: lytic or lysogenic. In the lytic mode, almost all of the phage genes are transcribed and translated, and the phage DNA is replicated, leading to production of progeny phages and lysis of the host cells. In the lysogenic mode, the lambda DNA is incorporated into the host genome; after that occurs, only one gene is expressed. The product of this gene, the lambda repressor, prevents transcription of all the rest of the phage genes. However, the incorporated phage DNA (the prophage) still replicates, since it has become part of the host DNA.

Figure 8.12 Lytic versus lysogenic infection by phage λ. Blue cells are in the lytic phase; yellow cells are in the lysogenic phase; green cells are uncommitted.

SummarySummary

Figure 8.13 Genetic map of phage lambda. (a) The map is shown in linear form, as the DNA exists in the phage particles; the cohesive ends (cos) are at the ends of the map. The genes are grouped primarily according to function. (b) The map is shown in circular form, as it exists in the host cell during a lyric infection after annealing of the cohesive ends.

Lytic Reproduction of λ PhageLytic Reproduction of λ Phage The immediate early/delayed early/late transcriptional switching in the lytic cycle of phage lambda is controlled by antiterminators. One of the two immediate early genes is cro, which codes for a repressor of the cI gene that allows the lytic cycle to continue. The other, N, codes for an antiterminator, N, that overrides the terminators after the N and cro genes. Transcription then continues into the delayed early genes. One of the delayed early genes, Q, codes for another antiterminator (Q) that permits transcription of the late genes from the late promoter, PR', to continue without premature termination.

Figure 8.14 Temporal control of transcription during lytic infection by

phage lambda. (a) Immediate early transcription (red) starts at the rightward and leftward promoters (PR‘ and PL, respectively) that flank the repressor gene (cI); transcription stops at the rho-dependent terminators (t) after the N and cro genes.(b) Delayed early transcription (blue) begins at the same promoters, but bypasses the terminators by virtue of the N gene product. N. which is an antiterminator.(c) Late transcription (gray) begins at a new promoter (PR'); it would step short at the terminator (t) without the Q gene product, Q, another antiterminator. Note that O and P are protein- encoding delayed early genes, not operator and promoter.

Figure 8.15 Effect of N on leftward transcription.

(a) Map of N region of λ genome. The genes surrounding N are depicted, along with the leftward promoter (PL) and operator (OL), the terminator (red), and the nut site (green). (b) Transcription in the absence of N. RNA polymerase (pink) begins transcribing leftward at PL and stops at the terminator at the end of N. The N mRNA is the only product of this transcription (c) Transcription in the presence of N. N (purple) binds to the nut region of the transcript, and also to NusA (yellow), which, along with other proteins not shown, has bound to RNA polymerase. This complex of proteins alters the polymerase so it can read through theterminator and continue into the delayed early genes.

Figure 8.16 Protein complexes involved in N-directed antitermination. (a) Weak, non-processive complex. NusA binds to polymerase, and N binds to both NusA and box B of the nut site region of the transcript, creating a loop in the growing RNA. This complex is relatively weak and can cause antitermination only at terminators near the nut site (dashed arrow). These conditions exist only in vitro. (b) Strong, processive complex. NusA tethers N and box B to the polymerase, as in (a); in addition, S10 binds to polymerase, arid NusB binds to box A of the nut site region of the transcript. This provides an additional rink between the polymerase and the transcript, strengthening the complex. NusG also contributes to the strength of the complex. This complex is processive and can cause antitermination thousands of base pairs downstream in vivo (open arrow).

Figure 8.17

Figure 8.18

Figure 8.19

Figure 8.20 Map of the PR' region of the λ, genome. The PR‘ promoter comprises the -10 and -35 boxes. The qut site overlaps the promoter and includes the Q binding site upstream of the -10 box, the pause signal downstream of the transcription start site, and the pause site at positions +16 and +17.

5 proteins (N, NusA, NusB,5 proteins (N, NusA, NusB, NusG and S10) collaborate in NusG and S10) collaborate in

antitermination at theλ antitermination at theλ immediate early terminatorsimmediate early terminators. .

NusA and S10 bind to RNA polymeraseNusA and S10 bind to RNA polymerase N and NusB bind to the boxB and boxA N and NusB bind to the boxB and boxA

regionsregions N and NusB bind to NusA and S10N and NusB bind to NusA and S10 NusA stimulates termination by NusA stimulates termination by

interfering with the binding between interfering with the binding between upstream part of the RNA hairpin and the upstream part of the RNA hairpin and the core polymerasecore polymerase

N helps NusA bind RNA, preventing N helps NusA bind RNA, preventing hairpin formationhairpin formation

Establishing LysogenyEstablishing Lysogeny

The delayed early genes help establish The delayed early genes help establish lysogeny in two ways:lysogeny in two ways:

Some of the delayed early gene Some of the delayed early gene products are needed for integration products are needed for integration of the phage DNA into the host of the phage DNA into the host genome;genome;

The products of the The products of the cIIcII and and cIII cIII genes genes allow transcription of the allow transcription of the cIcI gene and gene and therefore production of the therefore production of the λrepressor.λrepressor.

The promoter used for The promoter used for establishment of losogeny is establishment of losogeny is PPRERE, which lies to the right of P, which lies to the right of PRR and and crocro. Transcription from . Transcription from this promoter goes leftward this promoter goes leftward through the through the cIcI gene. The gene. The delayed early genesdelayed early genes cII cII and and cIII cIII also participate in this also participate in this process: CII, by directly process: CII, by directly stimulating polymerase stimulating polymerase binding to Pbinding to PRERE and P and PII; CIII, by ; CIII, by slowing degradation of CII. slowing degradation of CII.

Figure 8.21 Establishing lysogeny. Delayed early transcription from PR gives cII mRNA that is translated to CII (purple). CII allows RNA polymerase (blue and red) to bind to PRE and transcribe the CI gene, yielding repressor (green).

Figure 8.22 Binding of CII at the -35 box of both PRE and PI promoters of λ, phage.

Ptashne and colleagues performed a DNase footprint analysis of the interaction between CII and two early λ. promoters, PRE (a) and PI (b), In (a), lanes 1-4 contained the following amounts of CII: lane 1, none; lane 2, 10 pmol; lane 3, 18 pmol; and lane 4, 90 pmol. In (b), lanes 1-4 contained the following amounts of CII: lane 1, none; lane 2, 18 pmol; lane 3, 45 pmol; lane 4,100 pmol. The CII footprint in both promoters includes the -35 box.

Figure 8.23

Summary Summary

Phage λ establishes lysogeny by causing Phage λ establishes lysogeny by causing production of enough repressor to bind to the production of enough repressor to bind to the early operators and prevent further early RNA early operators and prevent further early RNA synthesis. The promoter used for establishment synthesis. The promoter used for establishment of lysogeny is of lysogeny is PPRERE, which lies to the right of , which lies to the right of PPRR and and crocro. Transcription from this promoter goes . Transcription from this promoter goes leftward through theleftward through the cI cI gene. The products of the gene. The products of the delayed early genes delayed early genes cIIcII and and cIIIcIII also participate in also participate in this process: CII, by directly stimulating this process: CII, by directly stimulating polymerase binding to polymerase binding to PPRERE; CIII, by slowing ; CIII, by slowing degradation of CII.degradation of CII.

Autoregulation of Autoregulation of cIcI Gene During Gene During LysogenyLysogeny

Repressor turns off Repressor turns off interrupting lytic circleinterrupting lytic circle

PPRMRM activating repressor activating repressor synthesissynthesis

OORR controls leftward transcription of controls leftward transcription of cIcI

OORR1+O1+ORR2 repressor 2 repressor

Figure 8.24 Maintaining lysogeny. (bottom) Repressor (green, made originally via transcription from PRE) forms dimers and binds cooperatively to OR1 and 2. The protein-protein contact between repressor on OR2 and RNA polymerase (red and blue) allows polymerase to bind to PRM and transcribe cI. (top) Transcription (from PRM) and translation of the cI mRNA yields a continuous supply of repressor, which binds to OR and OL and prevents transcription of any genes aside from cI.

Figure 8.25 Map of the DNA fragment used to assay transcription from cI and cro promoters.

The numbers denote the distances (in bp) between restriction sites. The red arrows denote the in vitro cI and cro transcripts.

Figure 8.26 Analysis of the effect of λ repressor on cl and cro transcription in vitro.

Ptashne and colleagues performed run-off transcription (which actually produced "stutter" transcripts) using the DNA template depicted in, Figure 8.25. They included increasing concentrations of repressor as shown at bottom. Electrophoresis separated the cl and cro stutter transcripts, which are identified at right. The repressor clearly inhibited cro transcription, but it greatly stimulated cl transcription at low concentration, then inhibited cl transcription at high concentration.

Figure 8.27

Figure 8.28 Principle of intergenic suppression to detect interaction

between λ repressor and RNA polymerase.

(a) With wild-type repressor and polymerase, the two proteins interact closely, which stimulates polymerase binding and transcription from PRM. (b) The repressor gene has been mutated, yielding repressor with an altered amino acid (red). This prevents binding to polymerase. (c) The gene for one polymerase subunit has been mutated, yielding polymerase with an altered amino acid (represented by the square cavity) that restores binding to the mutant repressor. Since polymeraseand repressor can now interact, transcription from PRM is restored.

Summary Summary

Figure 8.29 Selection for intergenic suppressor of λ cI pc mutation. Susskind and colleagues used bacteria with the chromosome illustrated (in smalr part) at bottom. The chromosome included two prophages: (1) a P22 prophage with a kanamycin resistance gene (yellow) driven by a λ PRM promoter with adjacent λ OR; (2) a λ prophage containing the λ cI gene (light green) driven by a weak lac promoter. Into these bacteria, Susskind and colleagues placed plasmids bearing mutagenized rpoD (σ factor) genes (light blue) driven by the lac UV5 promoter. Then they challenged the transformed cells with medium containing kanamycin. Cells transformed with a wild type rpoD gene, or with rpoD genes bearing irrelevant mutations. could not grow in kanamycin. However, cells transformed with rpoD genes having a mutation (red X) that compensated for the mutation (black X) in the cI gene could grow This mutation suppression is illustrated by the interaction between the mutant σ factor (blue) and the mutant repressor (green), which permits transcription of the kanamycin resistance gene from PRM.

Figure 8.30 Activation by contacting σ. The activator (e.g., λ repressor) binds to an activator site that overlaps the weak -35 box of the promoter. This allows interaction between the activator and region 4 of σ, which would otherwise bind weakly, if at all, to the -35 box. This allows the polymerase to bind tightly to a very weak promoter and therefore to transcribe the adjacent gene successfully.

SUMMARY SUMMARY Intergenic suppressor mutation studies show Intergenic suppressor mutation studies show that the crucial interaction between repressor that the crucial interaction between repressor and RNA polymerase involves region 4 of the and RNA polymerase involves region 4 of the σσ subunit of the polymerase. This polypeptide subunit of the polymerase. This polypeptide binds near the weak -35 box of binds near the weak -35 box of PPRMRM, which places , which places the the σσ region 4 close to the repressor bound to region 4 close to the repressor bound to OORR2. Thus, the repressor can interact with the 2. Thus, the repressor can interact with the σσ factor, recruiting RNA polymerase to the weak factor, recruiting RNA polymerase to the weak promoter. In this way, promoter. In this way, OORR2 serves as an activator 2 serves as an activator site, and site, and λλ repressor is an activator of repressor is an activator of transcription fromtranscription from P PRMRM. It stimulates conversion . It stimulates conversion of the closed promoter complex to the open of the closed promoter complex to the open promoter complex.promoter complex.

Determining the Fate of a Infection: Determining the Fate of a Infection: Lysis or LysogenyLysis or Lysogeny

Whether a given cell is lytically or lysogenically infected by phage λ depends on the outcome of a race between the products of the cI and cro genes. The cI gene codes for repressor, which blocks OR1, OR2, OL1, and OL2, turning off all early transcription, including transcription of the cro gene. This leads to lysogeny. On the other hand, the cro gene codes for Cro, which blocks OR3 (and OL3), turning off cI transcription. This leads to lytic infection. Whichever gene product appears first in high enough concentration to block its competitor's synthesis wins the race and determines the cell's fate. The winner of this race is determined by the CII concentration, whichis determined by the cellular protease concentration, which is in turn determined by environmental factors such as the richness of the medium.

Figure 8.31 The battle between cI and cro.

(a) cI wins. Enough repressor (green) is made by transcription of the cl gene from PRM that it blocks poLymerase (red and blue) from binding to PR and therefore blocks cro transcription. Lysogeny results. (b) cro wins. Enough Cro (purple) is made by transcription from PR that it blocks polymerase from binding to PRM and therefore blocks cl transcription. The lytic cycle results.

Lysogeny InductionLysogeny Induction When a lysogen suffers DNA damage, it induces the SOS response. The initial event in this response is the appearance of a co-protease activity in the RecA protein. This causes the repressors to cut themselves in half, removing them from the λ operators and inducing the lytic cycle. In this way, progeny λ phages can escape the potentially lethal damage that is occurring in their host.

Figure 8.32 Inducing the Figure 8.32 Inducing the λλ prophage. prophage.

(a) Lysogeny. Repressor (a) Lysogeny. Repressor (green) is bound to (green) is bound to OORR (and (and OOLL) ) and and cIcI is being actively is being actively transcribed from the transcribed from the PPRMRM promoter.promoter. (b) The RecA co-protease (b) The RecA co-protease (activated by ultraviolet light or (activated by ultraviolet light or other mutagenic influence) other mutagenic influence) unmasks a protease activity in unmasks a protease activity in the repressor, so it can cleave the repressor, so it can cleave itself. itself. (c) The severed repressor falls (c) The severed repressor falls off the operator, allowing off the operator, allowing polymerase (red and blue) topolymerase (red and blue) tobind to bind to PPRR and transcribe cro. and transcribe cro. Lysogeny is broken.Lysogeny is broken.