Transcription Mechanism in Prokaryotes

Introduction

Transcription is the process by which the genetic information in DNA is copied into RNA. In prokaryotes the process is rapid and streamlined because there is no nucleus — transcription and translation can occur simultaneously in the cytoplasm. The bacterial model organism Escherichia coli is the basis for most classical studies of transcription.

Basic principles

• Direction of synthesis: RNA is synthesized in the 5' → 3' direction using the DNA template in the 3' → 5' orientation.
• No primer required: RNA polymerase initiates RNA synthesis de novo (without a primer).
• Substrates: Ribonucleoside triphosphates (ATP, UTP, GTP, CTP). Energy is provided by cleavage of high-energy phosphate bonds.
• Single enzyme system: Prokaryotes use one major RNA polymerase to synthesize mRNA, rRNA and tRNA (in contrast to eukaryotes which have multiple polymerases).

Structure of prokaryotic RNA polymerase

Core enzyme (α₂ββ'ω)

• α (alpha) subunits (2 copies): Needed for assembly and interactions with regulatory proteins.
• β (beta): Active site—catalyses phosphodiester bond formation.
• β' (beta prime): Strong DNA-binding subunit.
• ω (omega): Structural/stabilizing role.

Sigma (σ) factor

The σ factor associates with the core enzyme to form the holoenzyme. σ provides promoter recognition and specificity. Different σ factors direct RNA polymerase to different sets of promoters (for example, σ⁷⁰ is the major housekeeping sigma factor in E. coli, while σ³² is used for heat-shock genes).

Holoenzyme = Core enzyme + σ factor

Promoter regions and promoter recognition

Promoters are DNA sequences upstream of the transcription start site (+1) that signal where transcription should begin.

Key promoter elements (common in bacteria)

• −35 region (approx. 35 bases upstream of +1). Consensus: TTGACA. Important for initial binding of holoenzyme.
• −10 region (Pribnow box) (approx. 10 bases upstream of +1). Consensus: TATAAT. AT-rich region where DNA melting and open-complex formation begin.
• +1 site — transcription start site; first nucleotide of the nascent RNA.

Strength of a promoter depends on how closely its sequence matches the consensus and the spacing between the −35 and −10 elements. Regulatory proteins and sigma factors also influence promoter strength and usage.

Stages of transcription

Transcription proceeds in three broadly defined stages: initiation, elongation and termination. Below we describe each stage in detail.

Initiation

1. Promoter recognition and closed complex formation: The holoenzyme (core + σ) binds to promoter DNA to form a closed complex where DNA is still double-stranded.
2. Open complex (transcription bubble): At the −10 (Pribnow) region, the DNA strands separate over ~12–17 bp to create an open complex. This melting is easier because the region is AT-rich.
3. Initiation of RNA synthesis: The first ribonucleotides are added at the +1 site. RNA polymerase starts RNA synthesis de novo — usually the first nucleotide is a purine (ATP or GTP).
4. Abortive initiation and promoter clearance: During early initiation the polymerase may synthesize short transcripts (2–9 nt) and release them — a phenomenon called abortive initiation. After synthesizing about 9–12 nucleotides the polymerase clears the promoter, σ dissociates (or rearranges), and the enzyme shifts into productive elongation.

Note:

The transition from initiation to elongation is a regulated step — many transcriptional controls act by affecting promoter binding, promoter clearance, or σ factor availability.

Elongation

• Processivity: After promoter clearance the core enzyme transcribes the gene at a steady rate (≈ 40–80 nt s⁻¹ in bacteria; typical textbook value ≈ 40 nt s⁻¹ for E. coli).
• Directionality: The enzyme moves along the template strand in the 3' → 5' direction while adding ribonucleotides to the RNA in the 5' → 3' direction.
• Transcription bubble and RNA–DNA hybrid: The transcription bubble is maintained as the polymerase moves — about 12–17 bp of DNA remain unwound and an RNA–DNA hybrid of ~8–9 bp exists within the enzyme.
• Coupling with translation: In prokaryotes, ribosomes attach to the nascent mRNA and begin translation even before transcription is complete (co-transcriptional translation). This coupling accelerates the cellular response to environmental signals.

Termination

Termination releases the RNA transcript and disassembles the transcription complex. Two major types occur in bacteria:

Rho-independent (intrinsic) termination

• Encoded by a terminator sequence in DNA: a GC-rich inverted repeat followed by a run of A residues on the DNA template.
• When transcribed, the RNA forms a stable hairpin (stem–loop) followed by a stretch of uracils (poly-U) in the RNA.
• The hairpin causes the polymerase to pause and the weak rU–dA base-pairing (weak RNA–DNA hybrid) facilitates dissociation of the transcript.
• This mechanism does not require additional proteins.

Rho-dependent termination

• Requires the Rho (ρ) protein, a hexameric, ATP-dependent RNA helicase.
• Rho binds to a rho-utilization site (rut) on the nascent RNA (these sites are rich in cytosine and lack strong secondary structure).
• Rho translocates along RNA, uses ATP to move, and catches up with the paused RNA polymerase. It then unwinds the RNA–DNA hybrid, releasing RNA and terminating transcription.
• Rho-dependent termination is often used to terminate transcription of certain operons and is regulated by sequence and kinetics of transcription.

Examples and regulatory contexts

The lac operon (brief overview)

The lac operon in E. coli is an inducible system that controls lactose metabolism. When lactose (or allolactose) is present it acts as an inducer by inactivating the lac repressor, allowing RNA polymerase to access the promoter and transcribe the structural genes lacZ, lacY and lacA. Transcription initiation and promoter strength are key control points in this operon.

The trp operon (brief overview)

The trp operon is a repressible operon that controls tryptophan biosynthesis. When tryptophan levels are high, the trp repressor binds corepressor (tryptophan) and attaches to the operator, preventing RNA polymerase from initiating transcription. The trp operon also uses an attenuation mechanism — a transcriptional termination mechanism that senses the level of charged tRNA^Trp and adjusts transcription prematurely when tryptophan is abundant.

Regulation of transcription

Regulation happens mainly at initiation and uses several tools:

• Sigma factor variation: Alternative σ factors redirect RNA polymerase to specific gene sets (e.g., stress-response genes).
• Repressors/activators: DNA-binding proteins that block or enhance RNA polymerase binding or promoter clearance.
• Attenuation: A mechanism in some operons (e.g., trp operon) where transcription termination is controlled by translation of a leader peptide and formation of RNA secondary structures.
• Feedback control: Metabolite levels (e.g., tryptophan) can act as co-repressors or co-activators modifying regulator activity.

Significance and applications

• Rapid response: Coupled transcription–translation enables bacteria to adapt quickly to environmental changes.
• Antibiotic targets: Many antibiotics target bacterial transcription (for example, rifampicin binds the β subunit of bacterial RNA polymerase and inhibits initiation; it is used to treat tuberculosis).
• Biotechnology: Understanding bacterial transcription is essential for designing expression systems used to produce recombinant proteins (e.g., insulin) in bacteria.

Comparison with eukaryotic transcription (summary)

Feature	Prokaryotes	Eukaryotes
Location	Cytoplasm	Nucleus
RNA polymerases	Single major type	Multiple (I, II, III, plus mitochondrial polymerase)
Initiation factors	σ factor	General transcription factors (e.g., TFIID, TFIIH)
RNA processing	Minimal (rare splicing)	Extensive (5' cap, intron splicing, 3' poly(A) tail)
Coupling with translation	Yes	No (separated by nuclear membrane)

Important terms (quick glossary)

• Holoenzyme: RNA polymerase core enzyme associated with σ factor.
• Promoter: DNA sequence that directs RNA polymerase to the transcription start site.
• Open complex: Region of unwound DNA where transcription begins.
• Transcription bubble: The local unwound region of DNA during elongation.
• Rut site: RNA sequence recognized by Rho protein for rho-dependent termination.
• Attenuation: A regulation mechanism where premature transcription termination controls expression.

Exam tips

• Always mention σ factor when describing initiation in prokaryotes — it is the hallmark difference from eukaryotic initiation.
• Draw and label the −35 and −10 promoter regions and the +1 start site in diagrams.
• For termination, clearly explain both rho-independent (hairpin + poly-U) and rho-dependent mechanisms and give the role of Rho protein.
• When asked to compare with eukaryotes, focus on location, number of polymerases, initiation factors, processing, and coupling with translation.