Initiation and Termination of Prokaryotic Transcription
Transcription is Catalysed by a DNA-Dependent RNA Polymerase:
Transcription is the process by which RNA is synthesized from a DNA template. This reaction is catalysed by DNA-dependent RNA polymerase, an enzyme that reads one strand of DNA (the template strand) and synthesizes a complementary RNA strand.
Only one strand of DNA is copied.
In each transcription event, only the template strand is read by RNA polymerase.
The coding strand has the same sequence as the RNA transcript.
This ensures directional and specific synthesis of RNA.
Synthesis starts de novo.
RNA polymerase does not require a primer to initiate synthesis.
This is possible because it can bind directly to promoter sequences on DNA and start RNA synthesis from the first nucleotide.
Synthesis occurs in the 5’ to 3’ direction.
Nucleotides are added to the 3’ hydroxyl end of the growing RNA chain.
One PPi (pyrophosphate) is released for each nucleotide polymerised.
For every nucleotide added, a nucleotide triphosphate (NTP) is added.
The release of PPi drives the polymerisation reaction forward, making transcription energetically favourable.
Reactions always leave a free 3’ OH to which further nucleotides (NTPs) are added.
Forms of RNA Polymerase:
RNA polymerase exists in different forms that are important for transcription initiation and regulation. Fractionation has helped identify the core enzyme and the holoenzyme.
Core Enzyme (Core Complex):
The core enzyme is responsible for the elongation of RNA once transcription has started.
It is composed of multiple subunits (in prokaryotes, typically α₂ββ’ω).
The core enzyme remains largely unchanged regardless of the gene being transcribed.
It cannot initiate transcription alone; it requires additional factors for promoter recognition.
Holoenzyme (Complete Enzyme):
The holoenzyme consists of the core enzyme plus a sigma (σ) factor.
The sigma factor is essential for recognising promoter sequences on DNA, which allows transcription to initiate accurately.
Once elongation begins, the sigma factor may dissociate, leaving the core enzyme to continue RNA synthesis.
Sigma Factors:
Regulatory subunits that guide RNA polymerase to specific promoters.
Different sigma factors can recognise different sets of genes, allowing bacteria to respond to environmental changes.
E.g. in E.Coli, σ70 is the housekeeping sigma factor, while σ32 is involved in heat-shock response.
CTD - C-Terminal Domain of Alpha Subunit:
The C-Terminal Domain of the alpha subunit plays a role in interactions with transcriptional activators and other regulatory proteins.
It helps recruit RNA polymerase to promoters and can influence transcription efficiency.
The modularity of RNA polymerase (core + sigma) allows bacteria to transcribe different genes under different conditions without needing a completely different enzyme.
Different sigma factors “program” the enzyme to recognize different promoter sequences, so the right genes are transcribed at the right time.
Initiation of Transcription in Bacteria:
RNA Polymerase binds to DNA.
The bacterial RNA polymerase holoenzyme first binds non-specifically to double-stranded DNA.
The σ factor (e.g. σ70) allows the holoenzyme to scan along the DNA, recognising specific promoter sequences upstream.
Once a good match is found, the enzyme binds tightly to form a closed promoter complex.
It locates the promoter.
Promoters are directional - they define the transcription start site (+1), and which strand will serve as the template in the 3’ to 5’ direction.
The main consensus elements for σ70-dependent promoters are:
-35 region: TTGACA (initial σ contact and docking).
-10 (Pribnow) box: TATAAT (melting region).
The optimal spacing between there motifs (~17bp) ensures the correct polymerase orientation.
σ70 contacts both motifs simultaneously, positioning the polymerase for strand opening.
It separates the strands of DNA to gain access to the template (open complex formation).
RNA polymerase melts ~14bp of DNA around the -10 region.
This forms an open complex (transcription bubble), which exposes the template strand.
There is no helicase required - RNA polymerase itself has intrinsic helicase activity, driven by conformational changes and energy from NTP binding.
The σ subunit (particularly σ70 region 2.4) stabilises the melted DNA by binding the single-stranded template.
Initiates synthesis of RNA.
The enzyme begins synthesis de novo, without a primer.
The first nucleotide usually starts with a purine at the +1 position.
The first two NTPs pair with the template, forming the initial phosphodiester bond (requires Mg2+ at the active site).
Short transcripts of 2-9 nucleotides are produced and released, which is known as abortive initiation—this continues until a stable RNA-DNA hybrid forms.
❗This ‘trial-and-error’ allows the enzyme to test stability before full elongation.
Elongates the RNA (Promoter Escape).
Once the transcript reaches ~10 nucleotides (nt), the RNA polymerase undergoes a conformational change. The σ factor is released/loosened.
The enzyme clears the promoter and begins elongation.
The core enzyme (α₂ββ’ω) continues RNA synthesis, moving along DNA at ~40nt/s.
The transcription bubble moves with the enzyme, unwinding ahead and rewinding behind.
❗Although both DNA melting and unwinding involve disruption of hydrogen bonds between complementary bases, the terms differ in scale and mechanism.
Melting refers to the local strand separation that occurs during transcription initiation (∼14 bp around the –10 region), driven by conformational changes within RNA polymerase.
Unwinding describes the processive helicase-driven separation of strands during replication, which introduces topological strain and requires topoisomerase activity to relieve torsional stress.
Comparison of Transcription Initiation to DNA Replication Initiation:
Transcription:
RNA polymerase + σ-factor binds promoter (–35, –10 regions).
σ recognises the sequence, positions polymerase.
DNA melts (~14 bp) around the –10 (Pribnow) box — no helicase needed.
Starts RNA synthesis de novo (no primer).
Abortive transcripts (<10 nt) made before promoter escape.
Replication:
DnaA binds oriC (origin), opens AT-rich region.
DnaC loads DnaB helicase — unwinds DNA.
Primase (DnaG) lays down RNA primers.
DNA Pol III begins synthesis from primers.
Bidirectional forks form from origin.
Rho-Independent Termination (Intrinsic Termination):
Driven entirely by RNA secondary structure — no protein factor required.
Mechanism:
RNA polymerase transcribes a region containing an inverted repeat followed by a poly-U tract.
RNA folds into a GC-rich stem–loop (hairpin) structure.
The GC base pairs make the hairpin very stable.
The hairpin stalls the polymerase.
The following poly-U region forms weak U–A base pairs with the DNA template.
The weak hybrid dissociates and the RNA is released, leading to termination.
Rho-Dependent Termination:
Requires Rho factor (ρ), a hexameric RNA helicase powered by ATP.
Mechanism:
Rho binds to rut (Rho utilization) site on single-stranded RNA (rich in C, poor in G).
Rho moves 5′ → 3′ along RNA, using ATP hydrolysis.
When RNA polymerase pauses (e.g., at terminator regions), Rho catches up and pulls RNA out of the polymerase, disrupting the transcription complex.
Regulation insight:
Rho rarely terminates transcripts undergoing translation, as ribosomes physically block its access to RNA.
Hence, Rho termination mostly affects untranslated RNAs (like rRNA precursors or 3′ UTRs).
Transcription–Translation Coupling:
In bacteria, translation begins while transcription is still ongoing.
Ribosomes bind to nascent mRNA behind RNA polymerase.
Ribosomes prevent Rho loading, increasing transcriptional efficiency.
Experiments show coupling doubles polymerase velocity and reduces pausing.