lecture 26 - prokaryotic transcription

central dogma of molcular biology/pathway from DNA to protein

Gene Expression Levels

• Different gene products (usually proteins) are required:

  • In different cells, At different levels, At different times

• Cells regulate gene expression according to the needs of the moment

  • Mostly by controlling RNA production

• Genes can be expressed with different efficiencies

• Example (Gene A vs. Gene B):

  • Gene A is transcribed and translated much more efficiently than Gene B

  • Result: Protein A produced in much greater quantities than Protein B

chemical structure of RNA vs DNA

Why RNA Can Base Pair with DNA and Itself

• free base pairing

• RNA is most often single-stranded

• But it must base pair (bp):

  • With DNA during transcription

  • With itself to fold into secondary structures

uracil vs. Thymine: Base Pairing

• Uracil lacks the methyl group seen in thymine

• This difference does not affect Watson-Crick base pairing with adenine

• Uracil still pairs with adenine via the same hydrogen bonding pattern as thymine

Conventional vs. Non-Conventional Base Pairing with RNA

• Short complementary stretches of RNA can fold using:

  • Conventional (c) base pairing — Watson-Crick

  • Non-conventional (nc) base pairing — non-Watson-Crick

• These pairings fold RNA into 3D structures

• Structure is determined by nucleotide sequence

• 3D structures are more stable and can perform cellular functions

Comparison of eukaryotic and prokaryotic transcription

• Prokaryotes: transcription and translation are coupled — no separation in space or time

• Eukaryotes: transcription (+ processing) and translation are uncoupled — separated by the nuclear envelope

Overall strategies in regulating gene expression in prokaryotes

In bacteria, the concentration of a given mRNA dictates production of the protein it encodes. mRNA concentration depends on two factors:

• the rate of mRNA synthesis

• the rate of mRNA degradation

The three elements of gene control in prokaryotes are, in order of importance:

1. transcription initiation

2. RNA turnover (nuclease degradation)

3. transcription termination

RNA synthesis simple diagram & general info

The ssRNA “transcript” produced by transcription is complementary to the template strand, and shares the same nucleotide sequence as the coding strand, with the exception of having U in place of T. As with DNA synthesis, RNA synthesis occurs in the 5’→3’ direction.

directions in RNA synthesis

+1 : 5’ postion of RNA being made

upstream : to the left from +1, negative

downstream : to the right from +1

promoter sequence

• RNA pol binds to promoter sequences to initate RNA synthesis

how does RNA polymerase know where to bind

Sigma Factors Guide RNA Polymerase to Bacterial Promoters

RNA polymerase holoenzyme–promoter complex

• RNA polymerase holoenzyme = RNA core (α₂ββ'ω) + σ (sigma) subunit

• σ subunit's role: recognizes the promoter to initiate transcription

• σ factor binds directly at the:

  • −35 box (consensus: TTGACA)

  • −10 box (consensus: TATAAT)

• Transcription begins at the +1 site (mRNA start), just downstream of the −10 box

• RNA core + σ subunit = RNA polymerase holoenzyme, and σ subunit = promoter recognition (initiation)

how to map protein binding sites on DNA

DNase I footprinting

how does DNase I footprinting work

DNase I = a type of endonuclease that cuts unprotected DNA

Method:

• Two samples prepared:

  • DNA only (control)

  • DNA + protein (e.g., transcription factors bound)

• Both treated with DNase I under limiting reaction conditions (partial digestion)

• Protein bound to DNA "protects" that region from DNase I cleavage

• Protein is then removed and DNA denatured

• No cleavage occurs in regions of protein binding (the "footprint")

• Fragments separated by gel electrophoresis

Result (gel):

• DNA-only lane: cleavage products at every possible position → ladder of bands

• DNA + protein lane: a gap ("footprint") appears where the protein blocked DNase I access — missing bands correspond to the protein-protected region

RNA Polymerase Structure: Channels and Strand Movement

• DNA entry channel: where duplex DNA enters the enzyme

• RNA polymerase unwinds DNA to expose the template strand

• Template strand is read to synthesize RNA; the RNA-DNA hybrid formed here is an A-form helix (mix of RNA + DNA)

• Coding strand: displaced, non-template strand (re-anneals with template downstream)

• NTP entry channel: where incoming ribonucleotides enter to be added to the growing RNA

• Mg²⁺: required as a cofactor for catalysis (same general role as in DNA polymerase)

• RNA exit channel: where the newly synthesized RNA (5' end first) exits the enzyme

• DNA exit channel: where the DNA duplex re-forms and exits after transcription

• Overall direction of transcription: enzyme moves along DNA, synthesizing RNA 5'→3'

stages of transcription

1. initation

2. elongation

3. termination

Transcription Initiation

When is sigma factor released?

→ Sigma (σ) factor is bound to the RNA polymerase core enzyme (forming the holoenzyme) only during the initiation phase.

• σ recognizes and binds the promoter (−35 and −10 boxes), positioning the holoenzyme to begin transcription

• Once RNA polymerase has successfully begun synthesizing RNA and cleared the promoter (promoter clearance, early in elongation), σ is released

• The core enzyme (without σ) then continues elongation alone, synthesizing the rest of the RNA transcript

• σ can then be recycled and reused by a different core enzyme to initiate transcription elsewhere

Supercoiling During Transcription

• As RNA polymerase moves along DNA, it generates torsional stress:

  • Positive supercoils form ahead of the polymerase (direction of transcription)

  • Negative supercoils form behind the polymerase

• This happens because RNA polymerase must locally unwind DNA to access the template strand, but the DNA ends are fixed/constrained, so the unwinding twist gets pushed into supercoiling elsewhere

types of transcription termination in prokaryotes

• rho-dependent

• rho-independent

Rho-Independent Termination (brief)

• A GC-rich stem-loop (hairpin) forms in the RNA transcript

• Causes RNA polymerase to pause

• Followed by a weak dA-rU duplex (few H-bonds) → unstable → RNA transcript falls off on its own, no extra proteins needed

Rho-Dependent Termination (brief)

• Rho helicase binds a C-rich sequence on the RNA transcript

• Translocates along RNA (ATP-driven)

• Catches RNA polymerase → destabilizes RNA-DNA hybrid → releases transcript

a mechanism in bacteria where the Rho protein stops the synthesis of RNA by catching up to RNA polymerase and unwinding the RNA-DNA bond. It requires three key items: the Rho utilization (rut) site, a pause site, and ATP energy. [1, 2]

How the Process Works

• Binding to RNA: The ring-shaped hexameric Rho protein binds to a specific C-rich/G-poor sequence on the newly made RNA strand called the rut site (Rho utilization site). [1, 2]

• Movement along RNA: Using energy from ATP hydrolysis, the Rho protein moves along the RNA strand in the 5' to 3' direction, following behind the RNA polymerase. [1, 2]

• Catching the Polymerase: When the RNA polymerase temporarily stalls at a specific DNA sequence known as a pause site, the moving Rho protein catches up to it. [1]

• Release of RNA: Once it reaches the stalled enzyme, Rho uses its helicase activity to break the hydrogen bonds in the RNA-DNA hybrid, releasing the RNA molecule and ending transcription.

Rho-Independent Termination: Detailed Mechanism

• RNA polymerase pauses at a GC-rich sequence in the transcript (template is also GC-rich)

• The GC-rich RNA sequence base-pairs with itself, forming a stable hairpin near the 3' end (this folding = isomerization)

• The hairpin destabilizes the RNA:DNA hybrid

• GC-rich sequence is followed by ≥3 A's in the template → transcribed into U's in the RNA

• A-U pairing = only 2 H-bonds (vs. 3 for G-C) → weaker H-bonding in this region of the hybrid → further destabilization

• Combined effect (hairpin + weak A-U pairing): induces RNA to dissociate → transcription terminates

Sequence of events: Pause → Isomerize (hairpin forms) → Escape (weak UUU:AAA hybrid) → Terminate

rho-dependent terminators

• A terminator sequence at the end of the gene causes RNA polymerase to pause

• Upstream (5') of this, the RNA has a 70–100 nt CA-rich sequence called rut (rho utilization site)

• Rho (ρ) protein — a hexameric ATPase — binds RNA at the rut site

• Rho migrates 5'→3' along the RNA, hydrolyzing ATP, until it reaches the paused transcription complex

• Rho disrupts RNA-DNA base-pairing, releasing the RNA transcript

• Mechanism not fully understood — Rho functions like a helicase (unwinds the RNA:DNA duplex)