Bacterial Transcription Mechanisms: Sigma Factor Switching

• Bacterial transcription can undergo major shifts through three primary mechanisms.
• Sigma-factor switching.
• RNA polymerase switching.
• Antitermination.

Sigma Factor Switching

• Bacterial viruses, also known as phages, infect bacteria and take control of the host's transcription machinery.
• A time-dependent transcription program is established during the infection process.
• Early phage genes get transcribed first then later genes.
• Transcription of host genes ceases later in the infectious cycle, with phage genes exclusively transcribed.
• These shifts come from changes in the transcription machinery within RNA polymerase itself.
• Sigma (σ) factor determines the specificity of T4 DNA transcription.
• Shifting the transcription process requires σ.
• The process in B. subtilis and its phage, SPO1, has been studied.
• Like T4, SPO1 has a temporal transcription program and a large genome.

Temporal control of transcription in SPO1

• The temporal transcription program occurs as follows:
• Early genes expression within the first 5 minutes.
• Middle genes expression between 5-10 minutes.
• Late genes expression occurring after 10 minutes.
• Phage-encoded σ factors directs transcription switching, allowing association with the host core RNA polymerase.
• These σ factors alter the host polymerase specificity regarding promoter recognition, from early to middle to late stages.
• The host σ factor is specific for early phage genes.
• Phage gp28 protein switches specificity to middle genes.
• Phage gp33 and gp34 proteins switch the specificity to late genes.

RNA polymerase Switching

• Phages such as T7 possess smaller genomes and fewer genes than SPO1.
• These phages undergo three phases of transcription: Classes I, II, and III.
• The gene-1 is essential for class II and III gene expression within the five class I genes.
• Only class I genes are transcribed when gene-1 is mutated.
• Gene-1 codes for a phage-specific RNA polymerase that transcribes the T7 phage class II and III genes specifically.
• Host polymerase handles the transcription of class I genes.
• Gene-1(phage-polymerase), that transcribes the class II and III genes.

Antitermination: Infection of E. Coli by Phage λ

• Virulent phages kill host cells by lysing or breaking them open while replicating.
• Temperate phages infect cells without killing them.
• Temperate phage has two reproduction styles.
• Lytic mode: infection progression as in a virulent phage.
• Lysogenic mode: phage DNA integrates into the host genome.
• A 27-kD phage protein (λ repressor, cl) shuts down transcription of all genes, except for cl by binding to two phage operator regions
• Phage DNA integrates into the bacterial genome once lysogeny is established.
• A bacterium harboring integrated phage DNA (lysogen), contains integrated DNA known as a prophage.
• The phage DNA in the lysogen replicates along with the host DNA.
• The lytic reproduction cycle has three phases of transcription
• immediate early, delayed early and late, sequentially arranged on the phage DNA.

Genetic Map of Phage λ

• DNA exists in linear form in the phage.
• Phage DNA circularizes after infection of the host begins.
• The linear form has sticky ends.
• Transcriptional switches control gene transcription.
• Antitermination: A type of transcriptional switch used by phage λ.
• Host RNA polymerase transcribes the immediate early genes (cro and N) first.
• N gene product serves as an antiterminator that permits RNA polymerase to ignore terminators at the end of the immediate early genes.
• Same promoters are used for both immediate early and delayed early transcription.
• Another antiterminator (Q) permits transcription of the late genes from the late promoter to continue without premature termination when the late genes get transcribed.
• The genetic sites surrounding the N gene:
• Left promoters, PL.
• Operator, OL.
• Transcription terminator.
• N, when present, binds transcript of N-utilization site (nut site), interacts with a protein complex bound to polymerase and ignores normal transcription terminator and continues into delayed early genes.

Antitermination and Transcription: Two Immediate Early Genes

• cro
• Codes for a repressor of the cl gene.
• Allows lytic cycle to continue.
• N
• Codes for N
• An antiterminator

Protein complexes in N-directed antitermination

• In N-directed antitermination, protein complexes are important.
• Weak complex (NusA), strong complex (NusA, NusB, NusG, S10)
• Antitermination in the λ late region requires Q.
• Q binds to the Q-binding region of the qut-site as RNA polymerase is stalled just downstream of the late promoter.
• Binding of Q to the polymerase appears to alter the enzyme so it can ignore the terminator and transcribe the late genes.
• Phage establishes lysogeny by causing the production of repressor protein cl to bind to the early operators, preventing further early RNA synthesis.
• Delayed early gene products are used for integration.
• Integration into the host genome employing integrase.
• Products of cll and clll allow transcription of the cl gene and production of λ repressor.
• Promoter used to establish lysogeny is PRE.
• Delayed early transcription from PR produces cll mRNA that is translated to cll protein.
• CII allows RNA polymerase to bind to Pre and transcribe the cl gene, resulting in repressor transcription
• When λ-repressor binds as a dimer to λ-operator, it results
• turning off further early transcription and stimulating repressor's own synthesis by activating PRM.
• Interrupts the lytic cycle
• Turn off of cro is very important as the product Cro-protein acts to counter repressor activity

λ-Repressor Protein

• It is a dimer of two identical subunits.
• Each subunit has 2 domains with distinct roles.
• Amino-terminal is the DNA-binding end of the molecule.
• Carboxyl-terminal is site of repressor-repressor interaction, allowing dimerization.
• High levels of repressor can repress transcription from PRM.
• Process may involve the interaction of repressor dimers bound to OR1, OR2, and OR3.
• Repressor dimers bound to O₁1, O2, and O₁3 via DNA looping.
• The balance between lysis or lysogeny is delicate.
• Bacterial lawn with phage particles
• Lysogeny leads to infection and then cell death.
• Clear plaque is seen when 100% of cells undergo lysis.
• Turbid, or Lysogenized plaques are usually turbid, meaning live lysogen is present.
• Some infected cells suffer the lytic cycle, while others are lysogenized.

The Battle Between Cl and Cro

• The repressor blocks OR1, OR2, OL1, and O2 so turning off early transcription, leads to lysogeny.
• The Cro blocks Or3 and O₁3, turning off transcription, leads to lytic infection.
• Gene product in high concentration first determines cell fate.
• When lysogen suffers DNA damage and SOS response is induced the initial event is seen in a co-protease activity in RecA protein.
• Repressors are caused to cut in half, removing them from a operators (lytic cycle is induced).
• Progeny phage can escape potentially lethal damage occurring in the host.