Chapter 8 Major Shifts in Prokaryotic Transcription

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Chapter 8 Major Shifts in Prokaryotic Transcription. 8.1 Modification of The Host RNA Polymerase During Phage Infection. SPO1( B. subtilis phage, large DNA genome) Temporal program of transcription. - PowerPoint PPT Presentation

Transcript of Chapter 8 Major Shifts in Prokaryotic Transcription

  • Chapter 8 Major Shifts in Prokaryotic Transcription

  • 8.1 Modification of The Host RNA Polymerase During Phage InfectionSPO1(B. subtilis phage, large DNA genome)

    Temporal program of transcription

  • Figure 8.1 Temporal control of transcription In phage SPO1- infected B. subtilis. (a) Early transcription is directed by the host RNA polymerase holoenzyme, including the host factor (blue); one of the early phage proteins is gp28 (green), a new factor. (b) Middle transcription is directed by gp28, in conjunction with the host core polymerase (red); two middle phage proteins are gp33 and gp34 (purple and yellow, respectively); together, these constitute yet another factor. (c) Late transcription depends on the host core polymerase plus gp33 and 34.

  • Evidence for switching modelGenetic studies mutations in gene 28 prevent early-to-middle switch; mutations in gene 33 or 34 prevent middle-to-late switchBiochemical studies purification of RNA polymerase

  • Figure 8.2 Subunit compositions of RNA polymerases in SP01 phage-infected B. subtilis cells. Polymerases were separated by chromatography and subjected to SDS-PAGE to display their subunits. Enzyme B (first lane) contains the core subunits (', , and ), as well as subunit IV (gp28). Enzyme C (second lane) contains the core subunits plus subunits V (gp33) and Vl (gp34). The last two lanes contain separated and 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 is clearly competed best by middle RNA, and the product made by C plus 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 Transcription of phage SPO1 genes in infected B. subtilis cells proceeds according to a temporal program in which early genes are transcribed first, then middle genes, and finally late genes. This switching is directed by a set of phage-encoded factors that associate with the host core RNA polymerase and change its specificity from early to middle to late. The host is specific for the phage early genes; the phage gp28 protein switches the specificity to the middle genes; and the phage gp33 and gp34 proteins switch to late specificity.

  • 8.2 The RNA Polymerase Encoded in Phage T7T7 (E. coli phage, small genome)

    Temporal control of transcription in T7

    genesExpression stageproductClass IearlyPhage RNA polymerase, ect.Class IImiddleClass II proteinsClass IIIlateClass III proteins

  • Figure 8.4 Temporal control of transcription in phage T7-infected E. coil. (a) Early (class I) transcription depends on the host RNA polymerase holoenzyme, including the host factor (blue); one of the early phage proteins is the T7 RNA polymerase (green). (b) Late (class II and III) transcription depends on the T7 RNA polymerase.

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

  • 8.3 Control of transcription During 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 A and 6E. Losick and colleagues transcribed plasmid p213 in vitro with RNA polymerase containing A (lane 1) or E (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 A enzyme transcribed only the vegetative gene, while the E enzyme transcribed both the vegetative and sporulation genes.

  • Figure 8.8 Specificity of E determined by run-off transcription from the spollD promoter. Rong et al. prepared a restriction fragment containing the spollD promoter and transcribed it in vitro with B. subtilis core RNA polymerase plus E (middle lane) or B plus c (right lane) Lane M contained marker DNA fragments whose sizes are indicated at left The arrow at the right indicates the position of the expected run-off transcript from the spollD promoter (about 700 nt). Only the enzyme containing E made this transcript.

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

  • 8.4 Genes with Multiple PromoterThe B. subtilis spoVG GeneThe Anabaena Glutamine Synthetase GeneThe E. coli glnA Gene

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

  • Figure 8.10 Resolution of RNA polymerases that transcribe the spoVG gene from two different promoters. Losick and his colleagues purified polymerase from B. subtilis ceils that were running out of nutrients. The last purification step was DNA-cellutose 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 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 of a run-off transcription assay using a DNA fragment with two spoVG 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 (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 length. The column separated a polymerase that transcribed selectively from the downstream promoter and produced the shorter run-off transcript (fractions 19 and 20) from a polymerase that transcribed selectively from the upstream promoter and produced the longer run-off transcript (fractions 22 and 23).

  • Figure 8.11 Specificities of B and E. LOSiCk and colleagues purified sigma factors B and E by gel electrophoresis and testedthem with core polymerase by the same run-off transcription assay used in Figure 8.10. Lane 2, containing E, caused initiation selectively at the downstream promoter (P2). Lane 5, containing B, caused initiation selectively at the upstream promoter (P1). Lane 6, containing both factors caused initiation at both promoters. The other lanes were the results of experiments with other fractions containing neither factor.

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

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

  • 8.5 The E. coli Heat Shock GeneshtpR gene, 32 (H) Comparison of 32 and 70 gene:

    -35 sequence space -10 sequence70 TTGACA 16-18 TATAA32 CNTTGAA 13-15 CCCCATNT

  • SUMMARY The heat shock response in E. coli is governed by an alternative factor, 32 ( H) which displaces 70 ( A) and directs the RNA polymerase to the heat shock gene promoters. The accumulation of 32 in response to high temperature is due to stabilization of 32 and enhanced translation of the mRNA encoding 32 .

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