Molecular Biology: Chapter 6 — Transcription

6.1 Bacterial RNA polymerase #

The study of Bacterial RNA polymerase provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.

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6.1.1 Core enzyme and sigma #

The bacterial core is α2ββ′ω. The β and β′ subunits form the cleft and the RNA–DNA hybrid channel. It does not find promoters. coli* is σ70. Alternative sigmas reassign the same core: σ32 to heat shock, σS to stationary phase, σ54 to nitrogen metabolism. σ54 is the odd case. It recognises sequences near −24 and −12 and does not melt DNA until an activator hydrolyses ATP.

Sigma contacts the promoter, stabilises the open complex, and usually leaves once the polymerase has cleared the promoter. The elongating enzyme is the core. The first nucleotide keeps its 5′ triphosphate. The template is read 3′→5′, the RNA grows 5′→3′, and the transcript matches the non-template strand with U for T. coli* is about 20–80 nucleotides per second.

6.1.2 Promoters: −10, −35, and the UP element #

A typical σ70 promoter has two hexamers. The −10 element, or Pribnow box, has the consensus TATAAT. The −35 element has the consensus TTGACA. The spacer is about 17 bp, and sigma must touch both elements at once, so spacer length matters as much as the match. The lac promoter is poor on purpose and needs an activator (Chapter 10).

Some strong promoters, including ribosomal RNA promoters, add an UP element: an AT-rich stretch upstream of −35, roughly −40 to −60, contacted by the C-terminal domain of the α subunit. It is an extra contact, not a third copy of the −10 sequence. The consensus is a summary of many promoters, not a sequence that must be present base for base.

6.1.3 Initiation, elongation, and two modes of termination #

Holoenzyme binds as a closed complex, DNA still paired, then melts about positions −11 to +2 to form the open complex. While held at the promoter it often makes and releases abortive transcripts of up to about ten nucleotides. The elongation bubble is about 12–14 bp, with about eight or nine RNA nucleotides paired to the template.

Intrinsic, or rho-independent, termination needs no extra protein. The transcript folds a GC-rich hairpin followed by a run of U. The hairpin pauses the enzyme, and the weak rU–dA hybrid lets the RNA leave. Rho-dependent termination uses the hexameric helicase Rho, which binds C-rich rut sites and, using ATP, pulls the transcript out of a paused polymerase. A ribosome travelling close behind covers those sites. A nonsense mutation that releases the ribosome early can therefore expose a rut site and abort downstream genes of an operon. That effect is polarity, and it depends on the coupling described in Chapter 8.

6.2 Eukaryotic RNA polymerases #

The study of Eukaryotic RNA polymerases provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.

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6.2.1 What polymerases I, II, and III make #

Three nuclear RNA polymerases divide the work. The Roman numerals are not the DNA polymerase series.

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5S rRNA is not a pol I product. U6 is pol III, whereas the other spliceosomal snRNAs are pol II. Three polymerases are the animal case, not a rule for every eukaryote.

6.2.2 Pol II promoters, general transcription factors, and the CTD #

Pol II promoters are not a −10/−35 pair. Many genes have a TATA box, consensus TATAAA, centred about 25–30 bp upstream of the start. It is not universal.

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The general factors are TFIIA, B, D, E, F, and H. TFIID contains TATA-binding protein and TBP-associated factors. TFIIH opens the DNA with its helicase and phosphorylates the CTD with its kinase, CDK7.

The CTD is the repeat YSPTSPS on the largest subunit of pol II: about 52 repeats in humans and about 26 in yeast. Serine 5 phosphorylation is high near the promoter and recruits the capping enzyme. Serine 2 phosphorylation rises toward the 3′ end and recruits splicing and cleavage factors. Pol I and pol III transcripts are not capped, and those polymerases lack this domain.

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6.2.3 Enhancers and Mediator #

An enhancer increases transcription from a promoter. It may lie upstream, downstream, or in an intron, kilobases or sometimes hundreds of kilobases away, in either orientation. It does not send a substance along the DNA.

Mediator is the main bridge: about 20–30 subunits, depending on the species and on whether its kinase module is bound. It contacts activators on one side and the pol II machinery on the other. It is not a sequence-specific DNA-binding protein. Cohesin, the loop extruder of Chapter 2, helps arrange the contacts. Which activators are present is Chapter 11.

6.3 Key terms, summary, and self-check #

Key terms. Sigma; −10; −35; UP element; intrinsic terminator; Rho; pol I, II, III; TATA box; TFIIH; CTD; Mediator.

Summary. Bacterial holoenzyme is core plus sigma. σ70 reads a −10 and a −35 element about 17 bp apart, and an UP element can add α-subunit contacts. Initiation passes through closed and open complexes and abortive transcripts. Intrinsic terminators are a hairpin plus U; Rho terminates when ribosomes are not covering the RNA. RNA pol I makes the large rRNAs, pol II the mRNAs, and pol III the tRNAs and 5S rRNA. Pol II uses TATA or an initiator, and TFIIH both opens DNA and phosphorylates the CTD, serine 5 early and serine 2 late. Enhancers act by looping, with Mediator as the bridge.

Self-check.

1. What does sigma add that the bacterial core lacks, and which sigma needs an activator to melt DNA?

2. Where are the −10 and −35 elements, and what protein domain contacts the UP element?

3. What RNA structure is an intrinsic terminator, and why can a nonsense mutation cause Rho-dependent polarity?

4. Which polymerase makes 5S rRNA, which makes U6, and which makes the 28S precursor?

5. Which CTD phosphorylation recruits capping, and what does Mediator bind that is not a general transcription factor?