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
Describe promoter-centered regulation and its role in gene expression.
- Promoter-centered regulation involves factors that target the promoter DNA.
- TFs create a complex regulatory network coordinating RNAP distribution.
- Transcription initiation occurs within the context of the bacterial nucleoid, which can affect promoter activity
What are Class I and Class II activators in promoter-centered regulation?
Class I activators = bind upstream locations (-61, -71, -81, -91) and interact with αCTD of RNAP α subunit.
Class II activators =
- bind overlapping the promoter -35 region and interact with σ domain 4.
- can also make contacts with other parts of RNA polymerase, such as αNTD
How can DNA conformational changes and activator modulation lead to activation or repression of transcription?
- DNA conformational changes involve activators realigning promoter elements for RNAP binding
- Activator modulation involves repressors binding to activators and preventing their interaction with RNAP
- Repressors can suppress activator-dependent activation, leading to gene repression.
How does promoter escape regulation affect transcription?
- Promoter escape = last step of initiation before RNAP begins elongation
- Stronger promoter-polymerase interactions hinder promoter escape, potentially limiting transcription.
- Regulatory proteins can control promoter escape, influencing transcription initiation.
What is repression by roadblock, and how does it work?
-Repression by roadblock = involves DNA-binding proteins causing roadblocks for RNA polymerase
- Proteins interrupt RNA chain elongation, partially or completely
- Roadblocks can result from competition, occlusion, collision, sitting duck, or activator dislodgement mechanisms
How can DNA methylation influence transcription and regulatory interactions?
- Adenine methylation affects interactions between regulatory proteins and DNA.
- In E. coli, GATC methylation can enhance/inhibit transcription depending on the target gene
- Methylation can alter binding of proteins & transcription apparatus, leading to phase variation & gene regulation
How do riboswitches mediate transcriptional regulation?
- Riboswitches = segments of mRNA that bind small molecules, regulating protein production.
- In Listeria monocytoegenes - a vitamin B12 riboswitch controls pocR gene expression by regulating an overlapping asRNA
- In Clostridium acetobutylicum - T-box and S-box riboswitches control expression of ubiGmccBA operon
Explain the concept of transcriptional interference
- Transcriptional interference occurs when one RNA polymerase displaces another during elongation
- Coliphage 186 demonstrates this, where a strong promoter (pR) displaces a weaker promoter (pL).
- leads to down regulation of certain genes, while others continue expression
Describe the process of transcription attenuation and how it affects gene expression
- Transcription attenuation involves asRNA binding to mRNA, causing termination
- In Vibrio anguillarum, RNAβ binds to fatDCBA mRNA, causing termination after fatA gene
- results in differential expression of fatDCBA–angRT operon
How does DNA supercoiling influence gene regulation?
- Bacterial DNA maintained in a negatively supercoiled state.
- Supercoiling influenced by environmental stress & regulated by DNA topoisomerases
Explain the process of transcription termination and the different termination mechanisms
- Intrinsic termination involves a hairpin followed by a U stretch, leading to complex dissociation.
- Rho-dependent termination involves Rho binding to ribosome-free mRNA & using ATPase activity to displace RNA polymerase.
- Mfd-dependent termination involves Mfd recognizing stalled RNA polymerase & removing it from DNA
What is antitermination and how is it used in gene regulation?
- Antitermination allows RNA polymerase to read through terminators & continue transcription
- Used to regulate gene expression progression in phages & operons.
- Specific recognition sites required for antitermination factors to act
How is catabolite repression achieved in bacterial gene regulation?
- Catabolite repression (CCR) occurs when preferred carbon sources repress catabolic systems.
- Achieved through different regulatory mechanisms, like cAMP-CAP regulation and lactose/triptophan/arabinose-regulated promoters
