Unit 24 Question Bank
DNA-dependent RNA polymerase attaches at this specific site on the template strand; this is followed by initiation of RNA synthesis at the starting point, and the process continues until a termination sequence is reached.
The promoter determines two things: where transcription is to commence along the DNA, and how frequently this event is to occur.
In bacteria promoters are relatively simple — approximately 40 nucleotides long, with an eight-nucleotide-pair sequence about 35 bp upstream of the start and a six-nucleotide-pair A+T-rich sequence about 10 nucleotides upstream; the σ subunit helps the core enzyme recognize and bind to the promoter region.
In eukaryotes promoters are more complex, built from the TATA box, initiator sequence (Inr) and downstream promoter element (DPE), together with promoter-proximal elements (50–200 bp) and distal enhancers and repressors (1000–10⁵ bp). The TATA box has a particularly rigid requirement for both position and orientation, and is bound by TFIID through its TATA-binding protein (TBP) subunit.TMU Lecture 22 · Harper's ch.36, p.398 · 2019 and 2020/21 papers, Section I
It is catalysed by DNA-dependent RNA polymerase, the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene, and proceeds in three general steps: initiation, elongation and termination.
Key features: synthesis has 5′→3′ polarity, with the template strand read 3′→5′; substrates are ATP, GTP, CTP and UTP, U replacing T; and a primer is NOT involved, as RNA polymerases can initiate synthesis de novo. Only one strand — the template strand — is copied, and it will not necessarily be the same strand of the double helix for every gene.
Initiation: polymerase binds the promoter and a transcription “bubble” of 20 bp forms; the whole complex covers 30–75 bp. Elongation: 3′,5′-phosphodiester bonds are formed 5′→3′. Termination: at a termination sequence the transcript is released — in bacteria by an intrinsic terminator (hairpin plus AT stretch) or by rho, an ATP-dependent RNA-stimulated helicase.TMU Lecture 22 · Harper's ch.36
Position +1 is the transcript initiation site.
Upstream sequences constitute the promoter.
Downstream sequences comprise the introns and exons.
The RNA product is the primary transcript.
Do not confuse it with the promoter, which is only the initiation-signal part of it — the two are commonly set together, and the distinction is the mark.TMU Lecture 22
INTRONS are intervening sequences that interrupt the exons of mRNA-encoding genes and that neither appear in mature mRNA nor contribute to the genetic information ultimately translated into the amino acid sequence of a protein.
The intron RNA sequences are cleaved out of the transcript, and the exons of the transcript are appropriately spliced together IN THE NUCLEUS before the resulting mRNA molecule appears in the cytoplasm for translation.
Splicing depends on consensus sequences at the splice junctions and an internal branch site, and is performed by the spliceosome, assembled from snRNAs and snRNPs.
Alternative splicing provides for different mRNAs — one gene, several proteins — which is why the apparent wastefulness of introns is tolerated.TMU Lecture 22 · Harper's ch.36, p.404
1 · Capping. A 7-methylguanosine cap is added at the 5′ terminal. The 5′ cap is required both for efficient translation initiation and protection of the 5′ end from attack by 5′→3′ exonucleases.
2 · Polyadenylation. The mRNA is cleaved about 20 nucleotides downstream from an AAUAAA sequence, and poly(A) polymerase adds a tail subsequently extended to about 200 A residues. The poly(A) tail both protects the 3′ end from 3′→5′ exonuclease attack and facilitates translation. (Histone mRNA lacks a poly(A) tail.)
3 · Splicing. Introns are removed and exons are spliced together by the spliceosome.
The cap and poly(A) tail have a synergistic effect on protein synthesis: initiation factors bridge them, circularising the message — which also serves as a check that the transcript was completed and processed properly.TMU Lecture 22 · Harper's ch.36
Definition and general features
Transcription is RNA biosynthesis from a DNA template, catalysed by DNA-dependent RNA polymerase, the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene. Its products are mRNA, tRNA and rRNA.
Four features distinguish it from replication: it adheres to Watson-Crick base-pairing rules using ATP, GTP, CTP and UTP, U replacing T; it proceeds 5′→3′, reading the template strand 3′→5′; a primer is NOT involved, as RNA polymerases can initiate synthesis de novo; and it copies only the template strand — which is not necessarily the same strand of the helix for every gene.
The bacterial enzyme
Core enzyme α₂ββ′; holoenzyme α₂ββ′σ. α — assembly of the tetrameric core; β — ribonucleoside triphosphate binding site; β′ — DNA template binding region; σ — helps the core enzyme recognize and bind to the promoter region.
Initiation
The holoenzyme must bind DNA and locate a promoter — a search it performs by binding many regions of DNA with low affinity and scanning at ≥10³ bp/s until it recognizes a region it binds with higher affinity. E. coli has 4 × 10³ promoters in 4.2 × 10⁶ bp.
Then follows localized unwinding of the two strands to provide a single-stranded template — a transcription bubble of 20 bp, the whole complex covering 30–75 bp — and formation of phosphodiester bonds between the first few ribonucleotides of the nascent chain, which is attached to the polymerization site on the β subunit. After nucleotides 3 to ~10, the polymerase undergoes a conformational change and moves away from the promoter — PROMOTER CLEARANCE.
Elongation and termination
The polymerase catalyzes formation of 3′,5′-phosphodiester bonds in the 5′→3′ direction, using NTPs as building units.
Termination occurs by two mechanisms. Intrinsic terminators contain an inverted, hyphenated repeat followed by a stretch of AT base pairs; the inverted repeat, when transcribed into RNA, generates an RNA hairpin which causes RNA polymerase to pause, and the weak rU:dA hybrid then releases the transcript. Rho-dependent termination uses rho, an ATP-dependent RNA-stimulated helicase that disrupts the ternary transcription elongation complex composed of RNA polymerase, nascent RNA and DNA.
Comparison with eukaryotes
| Prokaryote | Eukaryote | |
|---|---|---|
| Polymerases | One, α₂ββ′ + σ | Three — Pol I (rRNA), Pol II (mRNA), Pol III (tRNA, 5S) |
| Promoter recognition | σ factor | General transcription factors (GTFs) |
| Promoter | ~40 nt; −35 and −10 boxes | TATA / Inr / DPE + proximal (50–200 bp) + distal (1000–10⁵ bp) elements |
| Primary transcript | Equivalent to the mRNA | Pre-mRNA — a precursor |
| Processing | None for mRNA | Cap, poly(A) tail, splicing, in the nucleus |
| Coupling | Translation begins before transcription ends | Separated by the nuclear membrane |
| Inhibitor | Rifampicin | α-Amanitin |
The eukaryotic complication
A complex consisting of 50 unique proteins provides accurate and regulatable transcription of eukaryotic genes. RNA polymerase II requires TFIIA, B, D (or TBP), E, F and H to facilitate promoter-specific binding and formation of the preinitiation complex (PIC); TFIID binds the TATA box through its TATA-binding protein subunit and consists of 15 subunits — TBP and 14 TAFs.
But RNA polymerase II and the GTFs can only catalyze basal or unregulated transcription in vitro. Regulated transcription additionally requires coactivators, Mediator, chromatin remodellers and chromatin modifying factors — because promoter accessibility, and hence PIC formation, is often modulated by nucleosomes, and nucleosome eviction by chromatin-active coregulators facilitates PIC formation and transcription.
Finally, eukaryotic pol II carries a carboxyl terminal repeat domain (CTD) of consensus Tyr-Ser-Pro-Thr-Ser-Pro-Ser, whose phosphorylation and dephosphorylation is critical for promoter clearance, elongation, termination, and even appropriate mRNA processing — the device that couples transcription to processing.
Why processing exists
The RNA molecules synthesized in mammalian cells are made as precursor molecules that have to be processed into mature, active RNA. In prokaryotes the primary transcript is equivalent to the mRNA molecule; in eukaryotes it is a pre-mRNA, and processing occurs primarily within the nucleus. The nuclear membrane is what makes this possible — a message a ribosome was already reading could not be spliced.
The processes of transcription, RNA processing, and RNA transport from the nucleus are highly coordinated, being cotranscriptionally coupled through the phosphorylated CTD of RNA polymerase II, which carries the processing machinery along with the enzyme.
1 · Capping the 5′ end
Mammalian mRNA molecules contain a 7-methylguanosine cap structure at their 5′ terminal. The 5′ cap is required both for efficient translation initiation and protection of the 5′ end of mRNA from attack by 5′→3′ exonucleases. Capping also occurs on snRNAs.
2 · Polyadenylation of the 3′ end
The mRNA is first cleaved about 20 nucleotides downstream from an AAUAAA sequence; poly(A) polymerase then adds a poly(A) tail, subsequently extended to about 200 A residues. The poly(A) tail both protects the 3′ end from 3′→5′ exonuclease attack and facilitates translation. Histone mRNA is the notable exception, lacking a tail.
Note the symmetry: each end is protected against the exonuclease that attacks from its own direction, and both promote translation. Moreover the cap and poly(A) tail have a synergistic effect on protein synthesis, because initiation factors bridge them and circularise the message — which also serves as a check that the transcript was completed properly.
3 · Splicing
Exons are the RNA sequences that appear in mature RNAs; introns are intervening sequences that neither appear in mature mRNA nor contribute to the genetic information ultimately translated. The intron RNA sequences are cleaved out and the exons appropriately spliced together in the nucleus before the mRNA appears in the cytoplasm.
Splicing requires consensus sequences at the splice junctions and an internal branch site, and is carried out by the spliceosome: pre-mRNA combines with the snRNPs and other proteins to form a spliceosome; within it, snRNA base-pairs with nucleotides at the ends of the intron; the transcript is cut to release the intron and the exons are spliced together; the spliceosome then comes apart, releasing mRNA which now contains only exons. The catalysis is performed by RNA, not protein — the snRNAs are ribozymes.
Alternative splicing provides for different mRNAs, so one gene may specify several proteins — the reason introns are worth their cost.
Other processing, and editing
- Both ribosomal RNAs and most transfer RNAs are processed from larger precursors — the 45S transcript is cleaved to give the 18S, 5.8S and 28S rRNAs, and tRNAs and rRNAs undergo base modifications.
- RNA editing — reactions that change the nucleotide sequence of an mRNA by non-splicing mechanisms; the change may include nucleotide change, deletion or insertion. The classic case: the mRNA for apolipoprotein B in the liver is translated to apo B-100, while in the small intestine the mRNA is changed to yield a new termination codon (UAA), resulting in a much shorter protein, apo B-48.
Errors or changes in synthesis, processing, splicing, stability or function of mRNA transcripts are a cause of disease.