Chapter 29:RNA Synthesis and Processing

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Eucaryotic pre-mRNAContains introns and exons.

TranscriptionTranscription is a DNA directed RNA synthesis.

Gene/cistron: A DNA segment that carries information for a protein, rRNA or tRNA….or a transcribed segment of DNA. There does not seem to be a consensus definition but excludes non-transcribed regions.

In procaryotes, polycistronic transcripts are common (1 promoter:several genes).

In eucaryotes, most mRNAs are monocistronic (1 promoter:1 gene).

TranscriptionOperon: a segment of DNA transcribed as a

single mRNA strand. It includes promoter and operator and is called a transcription unit (TU).

Promoter: a region of DNA where initiation occurs, unique for a given transcription unit, TU.

Operator: DNA sequence close to promoter that regulates procaryotic transcription.

Enhancer: Eucaryotic regulatory sequence, may be upstream or downstream from promoter, some are bidirectional.

Conventions

dsDNA: 5' ----------- top strand --------------3' 3' ----------lower strand -------------5'

upstream(-) downstream(+) there is no 0 (zero) in DNA numbering

Top strand = coding strand = sense strand (+) Bottom strand = template = anti-sense strand (-)

So, the RNA formed (+) has the same relative sequence as the DNA sense strand and is formed form the template strand.

RNA Polymerase

E.Coli RNA Polymerase

The core polymerase is: α2ββ'ω

The holoenzyme is: α2ββ'ωσ

ω is a subunit of unknown function.

Polymerase Subunits

E.Coli has a number of different σ subunits.

These seek out different promoters when bound to the core enzyme then bind to promoter to start initiation.

Binding: Kd t½

Holoenzyme + random DNA 10-7 ~3 sec Holoenzyme + promoter 10-14 ~2-3 hr Core enzyme + random DNA 10-12 ~60 min

Initiation

Holoenzyme binds loosely to ds DNA and tracks downstream until σ recognizes the promoter region. It then locks on to DNA to form a closed promoter complex.

There are two consensus sequences within the promoter region.

-10 TATA box (Pribnow box) 5'-TATAAT-3' -35 box 5'-TTGACA-3'

When the polymerase is bound, bubble opening occurs ~-9 to +3 forming an open promoter. Unwinding and synthesis of first residues is slow.

Promoter Regions

Consensus Sequence

Promoter Regions and Transcription Start Site

Bubble Opening

Elongation

RNA polymerase needs supercoiled, dsDNA and Mg++. NTPs are substrate and PPi is released at each polymerization step.

RNA is synthesized in the 5' to 3' direction and copies only one DNA strand while moving downstream.

The polymerase does not require a primer but does appear to have some exonuclease (proofreading) activity.

The transcription product has leader and trailer segments.

Elongation

Polymerization starts at +1. The first residue is usually a purine. After RNA polymerase clears the promoter region (~6-10 residues) the σ subunit dissociates. Elongation uses the core polymerase and moves ~ 50 bp/sec.

DNA continually unwinds ahead of the bubble and rewinds behind so the bubble moves remaining about the same size. Two topoisomerases are needed, one ahead and one behind to introduce and remove supercoils.

When polymerase is free of promoter, another initiation may begin.

Magnesium Complex

Looks very much like the Mg++ complex for DNA.

Polymerization

This process is similar to that for DNA.

5' Residue

The 5' end is usually pppA or pppG and retains the phosphates.

Schematic of Elongation

Termination - Hairpin

Hairpin: This is a pause site. A G:C rich, inverted repeat region of DNA prior to the termination sequence. The inverted repeats are separated by a few A:Ts. There is a short A:T region at the end of the transcription unit.

A hairpin is formed in the RNA transcript using the G:C inverted repeats (tight binding). The protein NusA facilitates pause at the termination site. The polyU segment subsequent to the hairpin is loosely held and permits dissociation from the DNA template.

Hairpin and PolyU Segments

The RNA transcript.

Termination - rho

Rho () factor: is a hexameric protein which also acts as an ATP dependent helicase.

It binds at a specific recognition sequence in the transcribed RNA (upstream of the termination site). then follows the strand 5'-3' until it catches the transcription bubble and then unwinds the RNA from the template using its helicase activity to end transcription.

Termination and Rho

The RNA transcript.

Rho Tracking on RNA

Post-Transcription

Procaryotes:

mRNA shows little or no post transcriptional modification.

rRNAs and tRNAs are cleaved from the primary transcript and modified.

E.coli has about 7 rRNA genes and about 60 tRNA genes that are not auxillary to an rRNA gene. About 30-40% of the bases in tRNA are modified by methylation or other. CCA is added to the 3‘ end of those tRNAs in which it was not coded using a nucleotidyl transferase, 2 CTP and 1 ATP.

E.Coli Primary transcript

RNase III cleaves 5S, 16S and 23S precursors and RNases M 5, M 16 and M 23 trim these to yield mature mRNAs (except for CCA).

RNase P cleaves the 5' end of pre-tRNAs, RNase E & F cleave the 3' ends and RNase D trims.

E.Coli Primary transcript

Methylation

Methylation in rRNA:

N6,N6-diMe Adenine

Modificationsto Uracil

Common in tRNA:

Methylation.

Moving ribose from N1 to C5.

Eucaryotic RNA Polymerases

All three polymerases require transcription factors to interact with promoters. These are DNA binding proteins that initiate transcription at a specific promoter sequence

Eucaryotic PromotersPromoter and other regulatory sites on the same DNA molecule as the gene to be transcribed are referred to as “cis acting” elements.

Pol I uses a ribosomal initiator element (rInr) which is TATA-like sequence. In addition it uses an upstream promoter element about 150 bp from the start site.

Eucaryotic PromotersPol II uses a set of conserved sequences at the start site. These may be a TATA box found with an initiator element (Inr) to define the start site. In absence of a TATA box a downstream promoter element works with Inr. Other elements including an enhancer which can be 1 kbp from the start site may be used.

Bases from 100 Promoters

Eucaryotic TATA boxes, consensus relationships.

Other Promoter ElementsRecognized by proteins other than polymerase.

Eucaryotic PromotersOther cis-acting elements observed with Pol II are a CAAT box and/or a GC box. Several transcription factors associate with Pol II and its functioning is regulated by phosphorylation of a carboxyl-terminal domain (CTD).

Pol III promoters are within the gene itself downstream of the start site and are different for tRNA and rRNA.

General - Regulation

Activator = positive regulator that binds to promoter and stimulates transcription.

Case I: ligand binds to activator. This prevents binding to promoter and transcription does not occur.

Case II: ligand binds to activator. Ligand enhances binding of activator promoter and transcription is enhanced.

General - Regulation

Repressor = negative regulator that binds to promoter and prevents transcription.

Case I: ligand (inducer) binds to repressor. This prevents repressor binding to promoter, so transcription occurs.

Case II: ligand (corepressor) binds to repressor. Repressor will not bind promoter without ligand and transcription does not occur.

Modification of Pre-rRNAEucaryotic pre-rRNA: note the different size rRNAs compared to procaryotic rRNA.

Modification of Pre-tRNAProcessing eucaryotic pre-tRNA

Modification of Pre-mRNA

Primary mRNA transcripts are anywhere from 2000 to 20000 residues and sometimes called heterogeneous nuclear RNA (hnRNA).

Processing eucaryotic pre-mRNA: 1. The 5' end is capped: Remove P, add GTP with loss of PPi. Protects 5' from exonuclease. 2. A poly(A) tail is added: Cleavage and poly adenylation specificity factor (CPSF) binds specific sequence, endonuclease activity cleaves downstream and dissociation occurs. Poly(A) polymerase adds 150-250 residues to 3'. 3. Splicing occurs: Remove introns, join exons.

5' Capping pre-mRNA

Cap 0 = N7-Me G at 5'

Cap 1 = N7-Me G at 5' + O2'-Me ribosyl at 2nd residue.

Cap 2 = N7-Me G at 5' + O2'-Me ribosyl at 2nd and 3rd residue.

rRNA and tRNA are not capped.

Poly A Tail

Splicing

Introns are intervening sequences. These vary with size and number. E.g. ovalbumin has 7 introns whereas procollagen has 50.

Exons are expressed sequences.

All introns have conserved 5'GU …………AG3' termini and a branching sequence (splice start site) between these ends. Both of the ends are in consensus sequences. In mammals, there is more variability in the branch sequence.

Splicing

O2' of A at branch site attacks P at 3' end of AG in the upstream exon. Cleavage occurs by transesterification. The 3' OH of the upstream exon is released and attacks P at the 3' end of the intron. This joins the two exons by a second transesterification reaction.

Cleavage by Branch Point

An alcohol attacks a phosphodiester bond producing a new phosphodiester and a new alcohol. No ATP is involved in these reaction steps.

LariatIntermediate

The lariat occurs here.

Splicing Steps

Splicing Requires snRNA

snRNAs combine with proteins to form snRNPs. Assembly and binding of these “snurps” as well as helicase activity does require ATP. snRNAs have 2,2,7-triMeG-pppN cap at the 5' end.

Use of snRNAsMature mRNAs are considerably smaller than the original pre-mRNA transcripts. Some are reduced in size as much as 75-90%

Detail of events with snRNAs

snRNPs, splicing factors & pre-mRNA constitute a “spliceosome”.

Proposed events of Pol II

Transcription and processing of pre-mRNA requires the carboxyl-terminal domain (CTD) of Pol II.

CTD brings in protein required for capping, splicing and polyadenylation. The sequential occurrence of these events depends on the state of phosphorylation of CTD.

Note that in some cases alternate splicing of pre-mRNA can generate different mature transcripts and eventually peptides.

End of Chapter 29

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition