RNA Synthesis, Processing & Modification

Thymine is simply 5-methyluracil. The methyl group is a tag marking the base as belonging in DNA.

It matters because cytosine spontaneously deaminates to uracil. If uracil were a normal DNA base, that damage would be invisible; because it is not, a repair glycosylase can recognise every uracil in DNA as an error and excise it. The methyl group is the difference between a repairable lesion and a silent mutation.

Harper's compares them on four axes: abundance, size, function and general stability.

rRNA is the most abundant and the most stable; mRNA is the least stable, which is exactly what a regulatory message should be — a signal that persisted indefinitely could not be switched off.

The cloverleaf, with four arms:

The acceptor arm — the 3′-CCA-OH terminus, the site of attachment of the specific amino acid.
The anticodon arm — consists of seven nucleotides and recognises the three-letter codon in mRNA.
The TψC arm — involved in binding of the aminoacyl-tRNA to the ribosomal surface at the site of protein synthesis.
The D arm — one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase.

tRNA is rich in nontraditional nucleotides introduced by post-transcriptional modification — methylated guanosines, pseudouridine (ψ), inosine and others.

RNA biosynthesis from a DNA template is called transcription. Its products are mRNA, tRNA and rRNA.

The synthesis of an RNA molecule from DNA is a complex process involving one of the group of RNA polymerase enzymes and a number of associated proteins. The general steps required to synthesize the primary transcript are initiation, elongation and termination.

A promoter is the DNA sequence to which RNA polymerase binds to initiate transcription of a gene.

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.

A transcription unit is the region of DNA that includes the signals for transcription initiation, elongation and termination.

Position +1 is the transcript initiation site.
Upstream sequences (negative numbers) — the promoter.
Downstream sequences — introns and exons.
The RNA product is the primary transcript.

The enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene.

Four features distinguish it from DNA polymerase, and all four are examinable:

1 · It adheres to Watson-Crick base-pairing rules, using ATP, GTP, CTP and UTP — U replacing T.
2 · It synthesises with 5′→3′ polarity, reading the template strand in the 3′→5′ direction.
3 · A primer is NOT involved in RNA synthesis, as RNA polymerases have the ability to initiate synthesis de novo.
4 · Initiation requires large, multicomponent initiation complexes.

Core enzyme: α₂ββ′. Holoenzyme: α₂ββ′σ.

Functions of the subunits:
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

The transcription “bubble” is 20 bp of DNA, and the entire complex covers 30–75 bp.

Mammalian cells possess three distinct nuclear DNA-dependent RNA polymerases.

Pol I — most rRNA
Pol II — mRNA and most snRNAs and miRNAs
Pol III — tRNA and 5S rRNA

α-Amanitin is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool — it blocks the translocation of RNA polymerase during phosphodiester bond formation. It distinguishes the three because they differ in sensitivity to it.

Initiation: RNA polymerase binds to the promoter of DNA, and then a transcription “bubble” is formed. The holoenzyme must bind DNA and locate a promoter; then comes localized unwinding of the two strands by RNA polymerase to provide a single-stranded template, and formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain. The unwound complex is the preinitiation complex (PIC).

Elongation: the polymerase catalyzes formation of 3′,5′-phosphodiester bonds in the 5′→3′ direction, using NTPs as building units. The nascent chain is attached to the polymerization site on the β subunit.

Termination: when the polymerase reaches a termination sequence on DNA, the reaction stops and the newly synthesized RNA is released.

RNA polymerase continues to incorporate nucleotides 3 to ~10, at which point the polymerase undergoes another conformational change and moves away from the promoter; this reaction is termed PROMOTER CLEARANCE.

Until it happens, the polymerase repeatedly makes and releases very short abortive transcripts. Promoter clearance is therefore the commitment step of transcription.

Intrinsic terminators. The predominant bacterial transcription termination signal contains an inverted, hyphenated repeat followed by a stretch of AT base pairs. The inverted repeat, when transcribed into RNA, generates a secondary structure — an RNA hairpin — which causes RNA polymerase to pause. The weak rU:dA hybrid that follows then lets the transcript fall off. About 50% of genes use an inverted palindrome plus poly-A.

Rho-dependent termination. Rho is an ATP-dependent, RNA-stimulated helicase that disrupts the ternary transcription elongation complex composed of RNA polymerase, nascent RNA and DNA. It interacts with the paused polymerase and induces chain termination.

TATA box and Inr: 30% · Inr alone: 30% · Inr and DPE: 25% · all three elements: 15%.

Note what this means: the TATA box is present in only a minority of genes. The textbook picture of “every eukaryotic promoter has a TATA box” is wrong.

cis-acting elements are DNA sequences on the same molecule as the gene they control — promoters, enhancers, silencers.

trans-acting factors are diffusible proteins that bind them — the transcription factors.

Transcription factors have two functional parts: DNA-binding domains (DBDs) and activation domains (ADs).

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 both facilitate promoter-specific binding of the enzyme and formation of the preinitiation complex (PIC). RNA polymerases I and III require their own polymerase-specific GTFs.

TFIID binds to the TATA box promoter element through its TATA-binding protein (TBP) subunit; TFIID consists of 15 subunits — TBP and 14 TBP-associated factors (TAFs).

Crucially: RNA polymerase II and the GTFs can only catalyze BASAL or UNREGULATED transcription in vitro. Regulated transcription needs more.

The coactivators, or coregulators, work in conjunction with the DNA-binding transactivator proteins to communicate with Pol II and the GTFs to regulate the rate of transcription.

Promoter accessibility, and hence PIC formation, is often modulated by nucleosomes: nucleosome eviction by chromatin-active coregulators facilitates PIC formation and transcription. The machinery therefore includes Mediator, chromatin remodellers and chromatin modifying factors alongside the polymerase and GTFs.

Eukaryotic pol II consists of 12 subunits. The largest carries, at its carboxyl terminus, a carboxyl terminal repeat domain (CTD) with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, repeated many times.

The CTD is a substrate for several enzymes, and CTD phosphorylation/dephosphorylation is critical for promoter clearance, elongation, termination, and even appropriate mRNA processing.

Mammalian mRNA molecules contain a 7-methylguanosine cap structure at their 5′ terminal.

The 5′ cap of the RNA transcript is required both for efficient translation initiation and protection of the 5′ end of mRNA from attack by 5′→3′ exonucleases.

Most mRNAs have a poly(A) tail at the 3′ terminal.

The mRNA is first cleaved about 20 nucleotides downstream from an AAUAAA sequence; poly(A) polymerase then adds a poly(A) tail, which is subsequently extended to about 200 A residues.

The poly(A) tail both protects the 3′ end of mRNA from 3′→5′ exonuclease attack and facilitates translation.

Note the exception worth knowing: histone mRNA lacks a poly(A) tail.

Processing — cleavage of the 45S rRNA precursor, and base modifications of tRNAs and rRNAs
Capping — mRNAs and snRNAs
Polyadenylation — mRNAs
Splicing — mRNAs, some tRNAs

Both ribosomal RNAs and most transfer RNAs are processed from larger precursors; the 45S transcript is cleaved to yield the 18S, 5.8S and 28S rRNAs.

The RNA sequences that appear in mature RNAs are termed EXONS.

In mRNA-encoding genes, exons are often interrupted by long sequences of DNA that neither appear in mature mRNA, nor contribute to the genetic information ultimately translated into the amino acid sequence of a protein molecule. These intervening sequences are termed INTRONS.

The intron RNA sequences are cleaved out of the transcript, and the exons 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 on an internal branch site.

The spliceosome is assembled from snRNAs (small nuclear RNAs, the U series) and snRNPs (small nuclear ribonucleoprotein particles).

1 · Pre-mRNA combines with the snRNPs and other proteins to form a spliceosome.
2 · Within the spliceosome, snRNA base-pairs with nucleotides at the ends of the intron.
3 · The RNA 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 majority of miRNAs are transcribed by RNA pol II into primary transcripts termed pri-miRNAs. These are cut by Drosha into hairpins, transported into the cytoplasm and cut by Dicer, and the product anneals to the 3′ untranslated region of the target mRNA and interferes with protein translation.

Three mechanisms: (a) promoting mRNA degradation directly; (b) stimulating CCR4/NOT complex-mediated poly(A) tail degradation; (c) inhibition of translation by targeting the 5′-methyl cap binding translation factor eIF4.

RNA interference is triggered by the Dicer ribonuclease, which generates short interfering RNAs (siRNAs) of 21–28 bp. These are used to degrade target RNA by the RNA-induced silencing complex (RISC), and protect the host from RNA viruses.

RNA editing refers to the reactions that can change the nucleotide sequence of an mRNA molecule by non-splicing mechanisms. The change may include nucleotide change, deletion or insertion.

The classic example: the mRNA for apolipoprotein B in the liver is translated to apolipoprotein B100, while in the small intestine the mRNA is changed to yield a new termination codon (UAA), resulting in a much shorter protein, apolipoprotein B48.