Regulation of Gene Expression in Bacteria and Their Viruses Chapter 11

Metabolic Pathways and Additional Levels of Regulation: Attenuation Gene order in the trp operon corresponds to reaction order in the biosynthetic pathway In bacteria, genes that encode enzymes that are in the same metabolic pathways are generally organized into operons

Two mechanisms for regulating trp operon expression Level of trp operon gene expression is governed by the level of trp 2 ways Global control When trp (levels) are high… gene expression is repressed by a repressor that binds to an operator trpR Attenuation (fine-tuned control) A second level of regulation in amino acid biosynthesis operons is attenuation of transcription mediated by the abundance of the amino acid and translation of a leader peptide.

1. Global Control

2. Attenuator Control The trp mRNA leader sequence contains an attenuator region and two trp codons Since tryptophan is one of the least abundant proteins, two trp codons following each other is highly unusual. Parts of mRNA leader from stem-loop structures that alternate between two confirmations based on trp presence Leader Sequence

Abundant tryptophan attenuates transcription of the trp operon Remember bacteria transcription and translation can occur simultaneously But translation stalls when trp is low allowing for transcription to proceed and the stem loop doesn’t form

Same regulation in other amino acid biosynthesis operons The translated part of the trp leader region contains two consecutive tryptophan codons, (b) the phe leader sequence contains seven phenylalanine codons, and (c) the his leader sequence contains seven consecutive histidine codons.

Bacteriophage Life Cycles: More Regulators, Complex Operons The life cycle of bacteriophage λ Clear and cloudy bacteriophage plaques on a lawn of E. coli host bacteria Whether bacteriophage λ enters the lytic cycle immediately or enters the lysogenic cycle depends on the availability of resources. The lysogenic virus inserts its genome into the bacterial chromosome, where it remains quiescent until conditions are favorable. Plaques are clear where host cell lysis has occurred; they are cloudy where cells have survived infection and continued to grow as a lysogen. https://www.youtube.com/watch?v=kYf_Sl8W3qY

Lytic vs. lysogenic Depends on state of bacteria and resources If resources are abundant – lytic If resources are limited –lysogenic Virus remains as a prophage Inert prophage can be induced by UV light Both states of growth are characterized by very distinct programs of gene expression Controlled by several DNA-binding regulatory proteins and a set of operator sites

The phage λ genome organization facilitates coordinate control

Analogous genes between λ and lac operon Clear mutants (c) analogous to I and O mutants in lac system cI, cII and cIII mutants were all unable to establish lysogeny (Go Lytic) cro mutant enter lytic cycle in lysogenized cells cI represses lytic growth and promotes lysogenic λ Cro encodes a repressor that represses lysogeny premitting lytic growth cI and cro are in competition

The phage λ genome organization facilitates coordinate control Upon infection RNA pol initiates transcription of both promoters PR – cro PL – N N is a positive regulator and an anti-terminator cII – encodes an activator that promotes cI expression cI is a λ repressor which prevents lytic growth Antiterminator – prevent RNA pol from terminating in regions it should Thus you get the expression of cIII and cII cII encodes an activator protein that binds to a site the promotes the transcription leftward from a different promoter Pre (promoter of the repressor establishment)

cII is therefore the molecular decision maker cII is unstable because of its sensitivity to bacterial proteases Remember proteases degrade proteins More protease are active when resources are available (healthy) but less active when cells are starved So in the presence of resources, there is an increase in proteases which degrades cII, therefore cI can not be expressed and you have lytic growth cII also helps with int expression, an integrase, needed for integrations of the λ genome into the host chromosome cIII also shields cII from degradation so it too contributes to the decision.

Molecular anatomy of the genetic switch The lysogenic-versus-lytic cycle is determined by repressor occupancy on the OR operators Map of phage λ in the circular form. The genes for recombination, integration and excision, replication, head and tail assembly, and cell lysis are clustered together and coordinately regulated. Transcription of the right side of the genome begins at PR, and that of the leftward genes begins at PL. Key regulatory interactions governing the lysogenic-versus-lytic decision take place at operators between the cro and the cI genes. The phage λ genetic switch illustrates how a few DNA-binding regulatory proteins, acting through a few sites, control the expression of a much larger number of genes in the virus in a “cascade” mechanism. Just as in the lac, ara, trp, and other systems, the alternative states of gene expression are determined by physiological signals.

lytic state plasmid state lysogenic state plaque state Bacteriophage  enters the ________ by conservative site-specific recombination, during which time its genomic material is “integrated” into that of a host cell. lytic state plasmid state lysogenic state plaque state

Alternative Sigma Factors Regulate Large Sets of Genes Some responses to changes in the environment require the coordinated expression of large sets of unlinked genes For example: Sporulation Under stress, the bacterium forms spores that are remarkably resistant to heat and desiccation The mother cell and the forespore are produced from asymmetrically division of the parental cell Forespore -  F Mother cell -  E Both activate different gene sets - regulons

Alternative Sigma Factors Regulate Large Sets of Genes α factors control clusters of unlinked genes Sporulation in Bacillus subtilis is regulated by cascades of σ factors. (a) In vegetative cells, σA and σH are active. On initiation of sporulation, σF is active in the forespore and σE is active in the mother cell. These σ factors are then superseded by σG and σK, respectively. The mother cell eventually lyses and releases the mature spore. (b) Factors σE and σF control the regulons of many genes (ybaN, and so forth, in this illustration). Three examples of the large number of promoters regulated by each σ factor are shown. Each σ factor has a distinct sequence-specific binding preference at the −35 and −10 sequences of target promoters. Sequential expression of alternative σ factors that recognize alternative promoter sequences provides for the coordinated expression of large numbers of independent operons and unlinked genes during the developmental program of sporulation.

In Escherichia coli, the tryptophan operon encodes structural genes, including trpC, trpB, and trpA, that encode the tryptophan synthetase protein complex. The operon also encodes a repressor protein, encoded by the trpR gene. In the presence of high levels of tryptophan, tryptophan molecules bind the repressor protein and allow the repressor to recognize and bind to the operator, called trpO, and repress the expression of the structural tryptophan synthetase genes. In the absence of tryptophan, the repressor is unable to bind the operator and gene expression can occur. Assume any minus symbol represents a null mutant. What is the type of expression of each functional structural gene for the three bacterial strains below?

A scientist is culturing bacteria in media containing lactose, but no glucose. Which of the following statements is likely true of these cells? The concentration of cAMP in these cells will be high. In these cells, the CAP-cAMP complex will be bound to the lac operon. The lac operon will be transcribed in these cells. All of the answer options are correct.

The viral protein cII will be degraded in these cells. Bacteria are being cultured in resource-poor media containing bacteriophage . With these conditions, what will happen in these bacterial cells upon infection with phage ? Infected bacterial cells will lyse as a result of the phage entering the lytic cycle. The viral protein cII will be degraded in these cells. Two viral genes, cro and N, will NOT be transcribed. The viral gene cI will be transcribed (and NOT repressed).

Transcription of the arabinose or ara operon in bacteria is induced when a) only AraC is bound to two sites in the ara operon—araI and araO. b) two complexes are bound to araI in the arabinose operon: AraC-arabinose and CAP-cAMP. c) AraC (not attached to arabinose) is bound to the araO site in the ara operon, and CAP-cAMP is bound to the araI site. d) only the AraC protein is bound to the araI DNA sequence.

Imagine that you are studying sporulation in Bacillus subtilis, and have isolated a single specimen composed of a forespore and a mother cell. Which of the following statements will be true of this specimen? a) Different sigma factors will be present in the mother cell and forespore. b) The same type of RNA polymerase will transcribe different genes in the mother cell and forespore. c) One sigma factor, E, will be present in the mother cell but not the forespore. d) All of the answer options are correct.

The “ABC” operon encodes three enzymes: A, B, and C The “ABC” operon encodes three enzymes: A, B, and C. Upstream of the operon’s promoter, there is an independent gene that encodes the regulatory protein R. A bacterial strain from which the gene encoding R has been completely removed is found to transcribe the ABC operon even in the absence of an inducer. What can be concluded about this system? a) The ABC operon codes for enzymes that metabolize the inducer. b) The R protein binds to the inducer to allow transcription of the ABC operon. c) The R protein likely acts as a repressor of ABC operon transcription in the absence of an inducer. d) None of the answer options is correct.

The structures of target DNA sites Many DNA binding sites are symmetrical Can you see the two fold symmetry?