L6: Regulation of prokaryotic transcription Flashcards
How does transcription differ between prokaryotes and eukaryotes?
- Eukaryotic DNA in the nucleus transcribed by RNA polymerase (RNAP) in nucleus, then exported for translation.
- Prokaryotic DNA in the nucleoid in the cytoplasm, bound to histone-like proteins transcribed into mRNA by RNAP in the cytoplasm
- Both transcription and translation occur in the cytoplasm in prokaryotes
What constitutes a transcription unit and an operon in prokaryotes?
Transcription unit: Contains a 5’ non-template strand and a 3’ template strand; only template strand is used for mRNA production.
Operon: A cluster of genes regulated by a single promoter; includes promoter, operator, and RNA-coding region
What are the steps in prokaryotic transcription?
- Initiation: RNAP binds to promoter, DNA unwinds, RNA synthesis starts at the template strand.
- Elongation: RNAP moves downstream, unwinding DNA, elongating RNA transcript in the 5’–3’ direction.
- Termination: Terminator sequence is reached, RNA transcript is released, and RNAP detaches from DNA.
What is the function of σ factors in prokaryotic transcription?
- σ factors = TFs that bind to core RNAP to mediate transcription initiation
- target RNAP to specific promoters, melt promoter DNA & interact with other DNA-binding factors for gene expression regulation
How many types of σ factors are there in E. coli and what are their roles?
E. coli has seven σ factors:
σ70(σD) - Housekeeping
σ54(σN) - Nitrogen metabolism
σS - Stationary phase
σ32(σH) - Heat shock
σF(σ28) - Flagellar proteins
σE - Extreme heat shock
σfecl - Iron transport
What is the role of σ factors in E. coli transcription?
- They bind to core RNAP, helping it recognize specific promoters.
σ factors interact with promoter sequences around positions -10 and -35 or -12 and -24.
Each σ factor has a distinct role in gene regulation
Describe the cycle of RNA polymerase and σ factor interactions during transcription initiation
- Core RNA polymerase binds with a σ factor to form holoenzyme RNA polymerase.
- Holoenzyme binds to promoter sequence on DNA during initiation.
- After promoter binding, the σ factor dissociates from the holoenzyme.
- Core RNA polymerase continues transcription elongation and termination.
- After termination, core RNA polymerase can associate with another σ factor to initiate transcription of another gene
Explain the role of σ factors in transcription initiation and the open complex formation.
- σ factors guide RNA polymerase positioning at promoters & orchestrate open complex formation
- Housekeeping σ factor and alternative σ factors bind the same site on RNA polymerase.
- Housekeeping σ factor is abundant and outcompetes alternative σ factors.
- Housekeeping σ factor facilitates open complex formation, while alternative σ factors require ATP-dependent activators
What are the key DNA elements recognized by RNA polymerase at bacterial promoters?
UP element (positions -37 to -58)
-35 Box (positions -35 to -30)
Extended -10 element (positions -17 to -14)
-10 Box or TATA box (positions -12 to -7)
Discriminator element (positions -6 to -4)
Transcription start site (+1)
How are promoters categorized based on regulation and strength?
- Promoters can be constitutive, positively regulated, or negatively regulated.
- Constitutive promoters are regulated by RNA polymerase levels or sigma factors.
- Positively regulated promoters have increased activity with higher levels of an activator.
- Negatively regulated promoters have decreased activity with the presence of a repressor
What are the mechanisms that regulate RNA polymerase activity?
- Subcellular localization of RNA polymerase.
- Proteolytic turnover and limited proteolysis.
- Covalent modification of RNA polymerase.
- Rate of RNA polymerase synthesis.
- Sequestration of RNA polymerase.
- Presence of activators, repressors, operators, and inducers
Describe the role of σ factors and other regulators in redirecting RNA polymerase activity
- Some regulators (called appropriators) remodel RNA polymerase to alter promoter preferences.
- Examples: T4 phage AsiA and MotA proteins, phage T4 protein Alt, and E. coli SoxS.
- these factors can redirect RNA polymerase from host genes to phage genes or modulate promoter recognition based on stress conditions
How does the regulation of transcription initiation occur at the RNA polymerase-centered level?
- Factors can interact with RNA polymerase to influence its activity
- Factors include σ factors, other proteins & ligands that affect holoenzyme formation, activity, or promoter preferences.
- Some factors stabilize or destabilize open complexes, while others sequester RNA polymerase
How do fluctuations in nucleoside triphosphate (NTP) substrate levels affect RNA polymerase activity?
- RNAP activity can be regulated by changes in the levels of its NTP substrates
- Initiating NTP concentration important - especially for rRNA promoters.
- Higher levels of the initiating NTP (e.g., ATP) required for rRNA transcription due to its essential role in ribosome formation
Explain the role of anti-σ factors in transcription regulation.
- Anti-σ factors inhibit RNAP binding to σ factors.
- they stabilize σ factors in a conformation that prevents RNAP binding
- Anti-σ factors often have a modular structure with σ-binding and sensory/signaling domains.
- Co-transcription of anti-σ factor genes with σ factor genes helps maintain stoichiometric levels
