Chapter 8Chapter 8

Major Shifts inProkaryotic

Transcription

Modification of the Host RNA Modification of the Host RNA Polymerase Polymerase

• 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 σ factors that associated with the host core RNA polymerase and change its specificity from early to middle to late.

RNA polymerase changes specificityRNA polymerase changes specificity

• gp28: (1) diverts the host’s polymerase from transcribing host (2) switches from early to middle phage transcription gene

• gp33 and gp34: The switch from middle to late transcription occurs in much the same way, except that two polypeptides team up to bind to the polymerase core and change its specificity.

Fig. 8.1

• Genetic evidence: Mutants of gp28, gp34 or 33 prevent early-to-middle, middle-to-late switch

• Biochemical data: Pero measured polymerase specificity by transcribing SP01 DNA in vitro with core (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 the following competitors, early SP01 RNA (green); middle RNA (blue); and late RNA (red).

• Look for the competition for the products:

Control of Transcription During Control of Transcription During SporulationSporulation

• B. subtilis can exist indefinitely in the vegetative, as long as conditions are appropriate for growth.

• Under starvation conditions, this organism forms endospores, that can survive for years until favorable conditions return

• Sporulation is a fundamental change

Control of Transcription During Control of Transcription During Sporulation Sporulation

• 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 newσ factors that displace the vegetativeσ factor from the core RNA polymerase.

More than one new sigma factors are More than one new sigma factors are involved in sporulationinvolved in sporulation

• At least three sigma 29 (sigma E), sigma 30 (sigma H), and sigma 32 (sigma C) in addition to sigma 43 (sigma A) are involved.

The DNA region contains two promoters: a vegetative and a sporulation

In vitro transcription:Plasmid p213 + labeled nt+Sigma E or sigma A, then hybridized the labeled RNA to southern blot containing EcoRI-HincII fragments of the plasmid

Sigma E has some ability to recognize vegetative promoters

spoIID: well-characterizeSporulation gene.Rong prepared a restriction fragment containing the spoIID promoter and transcribed it in vitro with B. subtillis core RNA polymerase plus sigma E ( middle lane) or sigma B plus sigma C. Only the enzyme containing sigma E made the proper transcript.

Genes with Multiple Promoters Genes with Multiple Promoters

• Some prokaryotic genes must be transcribed under conditions where two differentσ factors are active. These genes contain two different promoters. This ensures their expression no matter which factor is present and allows for different control under different conditions.

Spo VG: transcribed by EB and E E.The last purification step was DNA-cellulose column chromatography. The polymerase activity in each fraction (red). The insert shows the results of a run-off transcription assay using a DNA with two SpoVG promoters.

Fig. 8.7

Purified sigma factors B and E by gel electrophoresis and tested them with core polymerase by the same run-off transcription assay.

Fig. 8.8

Fig. 8.9

The The E. coliE. coli Heat Shock Genes Heat Shock Genes

• When cells experience an increase in temperature, or a variety of other environmental insults, they mount a defense called the heat shock response.

• Molecular chaperones, proteases are produced.• At least 17 new heat shock transcripts begins

when at higher temperature (42 oC).• This shift of transcription required -32 (H).

Infection of E. coli by Phage Infection of E. coli by Phage

• Phage can replicate in either of two ways: lytic and lysogenic.

A bacterium harboring the integrated phage DNA is called a lysogenThe integrated DNA is called a prophage

Cro gene product blocks the transcription of repressor CIN: antiterminator

Extension of transcripts controlled by the same promoters. Q: antiterminator

Lytic reproduction of Phage Lytic reproduction of Phage

• The immediate early/delayed early/late transcriptional switching in the lytic cycle of phage is controlled by antiterminators.

N utilization site

NusA

N: function by restricting the pause time at the terminator

Antitermination Antitermination

• Five proteins (N, NusA, NusB, NusG and S10) collaborate in antitermination at the immediate early terminators.

• Antitermination in the late region requires Q, which binds to the Q-binding region of the qut site as RNA polymerase is stalled just downstream of the late promoter.

Highly conserved among Nut sites

Help to stabilize the antitermination complex

contains an inverted repeat

NusA, NusB, NusG, ribosomal S10 NusA, NusB, NusG, ribosomal S10 proteins interfere with antiterminationproteins interfere with antitermination

• Gel mobility shift assay: binding between N and RNA fragment containing box B

• NusA+ N bound to the complex: Fig. 8.16

Highly conserved among Nut sites

Help to stabilize the antitermination complex

contains an inverted repeat

Nus A and S10 bind to RNA polymerase, and N and Nus B bind to the box B and box A regions of the nut site in the growing transcript.

Fig. 8.15

Fig. 8.17

Qut: Q utilization siteQ binds directly to qut site not to the transcript

Establishing Lysogeny Establishing Lysogeny

• Phage establishes lysogeny by causing production of enough repressor to bind to the early operators and prevent further early RNA synthesis. The promoter used for establishment of lysogeny is PRE.

Fig. 8.18

Delayed early transcription from PR gives cII mRNA that is transcribed to CII (purple), which allows RNA polymerase (blue and red) to bind to PRE and transcribe the cI gene

Autoregulation ofAutoregulation of c cI Gene During I Gene During Lysogeny Lysogeny

• The promoter that is used to maintain lysogeny is PRM.

• It comes into play after transcription from PRE makes possible that burst of repressor synthesis that establishes lysogeny.

• This repressor binds to OR1 and OR2 cooperatively, but leave OR3 open. RNA polymerase binds to PRM,, in a way that contacts the repressor bound to OR2.

Fig. 8.19

Run-off transcription (this construct does not contain OL,

therefore, need to use very high concentration of repressor)

High levels of repressor can repress transcription from PRM, may involve interaction of repressor dimers bound to OR1, OR2 and OR3, with repressor dimers bound to OL1, OL2 and OL3 via DNA looping.

RNA polymerase-repressor RNA polymerase-repressor Interaction Interaction

• Intergenic suppressor mutation studies show that the crucial interaction between repressor and RNA polymerase involves region 4 of the σ subunit of the polymerase.

Fig. 8.23

Fig. 8.24

Fig. 8.25

Determining the fate of a Determining the fate of a Infection: Infection: lysis or lysogeny lysis or lysogeny

• Depends on the outcome of a race between the products of the cI and cro genes. The winner of the race is further determined by the CII concentration, which is determined by the cellular protease concentration, which is in turn determined by environmental factors such as the richness of the medium.

Fig. 8.26

Lysogen InductionLysogen Induction

• When a lysogen suffers DNA damage, it induces the SOS response.

• The initial event in this response is the appearance of a coprotease activity in the RecA protein.

• This causes the repressors to cut themselves in half, removing them from the operators and inducing the lytic cycle.

• In this way, progeny phages can escape the potentially lethal damage that is occurring in their host.

Fig. 8.27

Chapter 9Chapter 9

DNA – Protein Interactions in Prokaryotes

Helix 2 of the motif (red) lies in the major groove of its DNA target

9-51

The The Family of Repressors Family of Repressors

• Repressors have recognition helices that lie in the major groove of appropriate operator

• Specificity of this binding depends on amino acids in the recognition helices

9-52

Binding Specificity of Repressor-DNA Binding Specificity of Repressor-DNA Interaction SiteInteraction Site

• Repressors of -like phage have recognition helices that fit sideways into the major groove of the operator DNA

• Certain amino acids on the DNA side of the recognition helix make specific contact with bases in the operator

• These contacts determine the specificity of protein-DNA interactions

• Changing these amino acids can change specificity of the repressor

9-53

Probing Binding Specificity by Site-Probing Binding Specificity by Site-Directed MutagenesisDirected Mutagenesis

• Key amino acids in recognition helices of 2 repressors are proposed

• These amino acids are largely different between the two repressors

The helix-turn-helix motif of the upper monomer (red and blue) is inserted into the major groove of the DNA)

The repressor of the lambda-like phages have recognition helices that fit sideways into the major groove of the operator DNA.

Certain amino acids on the DNA side of the recognition helix make specific contact with bases in the operator, and these contacts determine the specificity of the protein-DNA interaction.

Changing these amino acids can change the specificity of the repressor.

9-57

High-Resolution Analysis of High-Resolution Analysis of Repressor-Operator InteractionsRepressor-Operator Interactions

• General Structural Features– Recognition helices of each repressor monomer

nestle into the DNA major grooves in the 2 half-sites

– Helices approach each other to hold the two monomers together in the repressor dimer

– DNA is similar in shape to B-form DNA– Bending of DNA at the two ends of the DNA

fragment as it curves around the repressor dimer

Fig. 9.6

General structural features

9-60

Interactions With BasesInteractions With Bases

9-61

Amino Acid/DNA Backbone InteractionsAmino Acid/DNA Backbone Interactions

• Hydrogen bond at Gln 33 maximizes electrostatic attraction between positively charged amino end of a-helix and negatively charged DNA

• The attraction works to stabilize the bond

The most important contacts occur in the major groove, where amino acids make hydrogen bonds with DNA bases and with the DNA backbone.

Some of these hydrogen bonds are stabilized by hydrogen-bond Networks involving two amino acids and two or more sites on the DNA.

Hydrogen bonds are represented by dashed lines, the van der Waals interaction between the Gln 29 side chain and the 5-methyl group of the thymine paired to adenine 3 is represented by concentric arcs

This implies hydrogen bonding between the protein and DNA at these sites.

This analysis also shows probable hydrogen bonding between three glutamine residues in the recognition helix and three base pairs in the repressor.

It also reveals a potential van der Waals contact between one of these glutamines and a base in the operator.

9-65

The Role of TryptophanThe Role of Tryptophan

• The trp repressor requires tryptophan to force the recognition helices of the repressor dimer into proper position for interacting with the trp operator

DNA deviates significantly from its normal regular shape.

It bends somewhat to accommodate the necessary base/amino acid contacts.

The central part of the helix is wound extra tightly.

Fig. 9.13

The trp repressor requires tryptophan to force the recognition helices of the repressor dimer into the proper position for interacting with the trp operator.

General considerations on Protein-DNA General considerations on Protein-DNA interactionsinteractions

• Specificity of binding between a protein and a specific stretch of DNA:

• 1. Specific interactions between bases and amino acids

• 2. the ability of the DNA to assume a certain shape, which also depends on the DNA’s base sequence.

9-71

Hydrogen Bonding Capabilities of the Hydrogen Bonding Capabilities of the Four Different Base PairsFour Different Base Pairs

• The four different base pairs present four different hydrogen-bonding profiles to amino acids approaching either major or minor groove

9-72

The Importance of Multimeric DNA-The Importance of Multimeric DNA-Binding Proteins Binding Proteins

• Target sites for DNA-binding proteins are usually symmetric or repeated

• Most DNA-binding proteins are dimers that greatly enhances binding between DNA and protein as the 2 protein subunits bind cooperatively

9-73

9.4 DNA-Binding Proteins: Action at a 9.4 DNA-Binding Proteins: Action at a DistanceDistance

• There are numerous examples in which DNA-binding proteins can influence interactions at remote sites in DNA

• This phenomenon is common in eukaryotes

• It can also occur in several prokaryotes

9-74

The The galgal Operon Operon

• The E. coli gal operon has two distinct operators, 97 bp apart– One located adjacent to the

gal promoter• External operator, OE

– Other is located within first structural gene, galE

• 2 separated operators -both bind to repressors that interact by looping out the intervening DNA

9-75

Effect of DNA Looping on DNase Effect of DNA Looping on DNase SusceptibilitySusceptibility

Operators separated by – Integral number of double-helical turns can loop out DNA

to allow cooperative binding

– Nonintegral number of turns requires proteins to bind to opposite faces of DNA and no cooperative binding

Fig. 9.17

9-77

EnhancersEnhancers

• Enhancers are nonpromoter DNA elements that bind protein factors and stimulate transcription– Can act at a distance– Originally found in eukaryotes– Recently found in prokaryotes

9-78

Prokaryotic Genes Can Use Prokaryotic Genes Can Use EnhancersEnhancers

• E. coli glnA gene is an example of a prokaryotic gene depending on an enhancer for its transcription

• Enhancer binds the NtrC protein interacting wit polymerase bound to the promoter at least 70 bp away

• Hydrolysis of ATP by NtrC allows formation of an open promoter complex

• The two proteins interact by looping out the DNA • Phage T4 late enhancer is mobile, part of the phage

DNA-replication apparatus

Fig. 9.18

Fig. 9.19

Fig. 9.20