Molgen-7 Prokaryotic Transcription
Course Information
Institution: Bahçeşehir University
Department: Molecular Biology and Genetics
Course: MBG2001 Molecular Genetics I
Lecture Title: Prokaryotic Transcription
Lecturer: Dr. Dilek ÇEVİK
Contact: dilek.cevik@bau.edu.tr
Copyright: © Bocos Benedict/Shutterstock. Copyright © 2018 by Jones & Bartlett Learning, LLC, an Ascend Learning Company
Website: www.jblearning.com
17.1 Introduction
Transcription Directionality:
Transcription occurs in a 5′ to 3′ direction.
Utilizes a DNA template that runs 3′ to 5′.
Coding (nontemplate) Strand:
The DNA strand that shares the same sequence with mRNA.
It is linked to the protein sequence it encodes through the genetic code.
RNA Polymerase:
An enzyme responsible for synthesizing RNA from a DNA template.
Formally referred to as DNA-dependent RNA polymerase.
Functional Role: Copies one strand of duplex DNA into RNA.
17.1 Additional Terminology
Promoter:
A specific region of DNA where RNA polymerase attaches to initiate transcription.
Start Point:
The position on DNA corresponding to the first base that gets incorporated into RNA.
Terminator:
A DNA sequence that signals RNA polymerase to stop transcription.
Transcription Unit:
The sequence between the initiation and termination sites transcribed into a single RNA molecule.
May include multiple genes.
17.1 Transcript Specific Terms
Upstream/Downstream:
Upstream: Sequences that are in the opposite direction of expression.
Downstream: Sequences advancing in the direction of expression within the transcription unit.
Primary Transcript:
The original RNA product that has not yet been modified and corresponds to a transcription unit.
17.2 Transcription Mechanism
Transcription Process Overview:
RNA polymerase creates a transient "bubble" by separating DNA strands.
Uses one strand (template strand) to guide the RNA synthesis.
Size of the Transcription Bubble:
Approximately 12 to 14 base pairs (bp).
RNA-DNA hybrid measures about 8 to 9 bp within the bubble.
17.3 Stages of the Transcription Reaction
Three Major Stages:
Initiation:
RNA polymerase binds to a promoter site on DNA forming a closed complex.
Conversion to an open complex occurs as transcription begins by unwinding the DNA.
Elongation:
The transcription bubble travels along the DNA.
The RNA chain is elongated in the 5′ → 3′ direction, adding nucleotides to the 3′ end.
Termination:
Transcription ceases as RNA polymerase separates from the DNA at a terminator site.
The DNA strands re-anneal.
17.4 Structure of Bacterial RNA Polymerase
Holoenzyme:
The functional form of RNA polymerase that initiates transcription.
Comprised of five subunits of the core enzyme and the sigma factor (σ).
Core RNA Polymerase Composition:
Multisubunit complex weighing approximately 400 kDa with the general structure α₂ββ′ω.
Catalytic Mechanism:
Catalysis occurs primarily through the β and β′ subunits.
C-Terminal Domain (CTD):
Part of RNA polymerase that stimulates transcription by interacting with regulatory proteins.
17.5 Holoenzyme Functionality
Sigma Factor Role:
The sigma factor is crucial for initiating transcription, modifying RNA polymerase's DNA-binding properties.
Reduces RNA polymerase's general DNA affinity but increases affinity for promoter regions.
17.6 Promoter Recognition and Binding
Understanding Binding Mechanics:
The binding rate of RNA polymerase to promoters is rapid, beyond simple diffusion.
RNA polymerase explores random DNA sites until it locates a promoter.
17.7 Holoenzyme Transitions in Promoter Interaction
Ternary Complex Formation:
Consists of RNA polymerase, DNA, and the first dinucleotide of the RNA product.
Abortive Initiations:
Repeated cycles may occur before transitioning to the elongation phase.
Release of Sigma Factor:
Typically occurs once the nascent RNA chain reaches around 10 bases in length.
17.8 Sigma Factor and Specific Promoter Sequences
Conserved Sequences:
DNA sequences with consistent nucleic acids across many examples at specific locations.
Consensus Sequences in Promoters:
Generally consists of:
Purine at the start point.
Hexamer sequence around TATAAT at –10 (TATA box).
Hexamer sequence similar to TTGACA at about –35.
Individual promoters may deviate from consensus sequences at one or more base positions.
Promoter Efficiency Enhancement:
Can be influenced by additional sequence elements, such as UP elements located upstream of the –35 element.
17.9 Mutation Effects on Promoter Efficiency
Down Mutations:
Decrease promoter efficiency by diverging from consensus sequences.
Up Mutations:
Enhance promoter efficiency through better adherence to consensus structures.
Mutations in the –35 sequence impact RNA polymerase's initial binding capabilities, while mutations in –10 can affect either binding or the transition from closed to open complex.
17.10 RNA Polymerase Interaction with Promoter DNA
Interactions Involving σ70:
Structural changes allow interaction with DNA-binding regions.
Multiple components of σ70 interact with promoter sequences, influencing transcription initiation.
17.11 Interaction Techniques
Footprinting Technique:
Identifies DNA-bound sites by assessing protection from nuclease activity.
Consensus Sequence Contacts:
The interactions between the sequences at –35 and –10 with RNA polymerase are critical for successful transcription initiation.
17.12 RNA Polymerase and Sigma Factor Recycling
Transcriptional Recycling:
The association of sigma factor and core enzyme is dynamic, allowing for efficient transcription processes.
17.13 Structural Model for RNA Polymerase Movement
Channel Dynamics:
DNA moves through a channel in RNA polymerase and experiences directional turns at the active site.
Flexible Module Role:
Modular structures within RNA polymerase provide flexibility, controlling nucleotide accessibility at the active site.
17.14 RNA Polymerase Pausing and Restarting
Transcription Pauses:
Certain DNA sequences can cause RNA polymerase pausing.
Restarting involves cleaving RNA transcript to establish a new 3′ end.
17.15 Termination of Transcription in Bacteria
Terminators:
Intrinsic Terminators: Recognized solely by RNA polymerase without other factors, marked by specific hairpin structures in RNA.
Rho-dependent Terminators: Require the rho protein for termination.
Termination Signals:
Located upstream of the terminator sequence, often within transcribed sequences, involving hairpin structures of 7 to 20 bp.
17.16 Rho Factor Mechanism
Functionality of Rho Factor:
Interacts with nascent RNA at the rut site (rho utilization site).
Tracks along RNA transcript to release RNA polymerase from the elongation complex.
Polarity effect: Mutation impacts expression of downstream genes.
17.17 Impact of Supercoiling on Transcription
Transcription Dynamics:
Negative supercoiling enhances promoter efficiency by aiding the melting reaction.
Transcription creates positive supercoils ahead and negative supercoils behind RNA polymerase requiring removal by gyrase and topoisomerase.
17.18 Models Using Phage T7 RNA Polymerase
T7 Family Significance:
Represents a valuable model system, recognizing phage promoters and mimicking multi-subunit RNA polymerases' activities.
Structural Insights:
Crystal structures depict the DNA-binding region and active site dynamics.
17.19 Competition among Sigma Factors**
E. coli Sigma Factors:
Total of seven sigma factors, each responsible for transcription initiation at distinct promoter sets defined by particular –35 and –10 sequences.
Regulation Mechanisms:
Variations in sigma factor activity controlled through different regulatory proteins such as anti-sigma factors.
17.20 Cascading Sigma Factor Organization
Cascade Functionality in Gene Regulation:
One sigma factor initiation allows subsequent sigma factors to be transcribed.
Example: Early genes of phage SPO1 regulate transcription via successive sigma factors.
17.21 Role of Sigma Factors in Sporulation
Sporulation Process:
Involves specialization of a bacterium into mother cell (that lyses) and a spore.
Contrast between vegetative phase (normal growth) and sporulation phase (spore formation).
17.22 Antitermination Mechanisms
Concept of Antitermination:
Antitermination complexes enable RNA polymerase to bypass terminators.
N Utilization Site (nut):
A sequence recognized by N antitermination factor in phage lambda's regulatory systems.