LECTURE 17: RNA TRANSCRIPTION, PROCESSING, TURNOVER

Levels of specific messenger RNAs can differ in different types of cellsand at different times in the same cell.

Control of RNA abundance can be at the level of transcription initiation,transcription elongation, processing or degradation.

RNA control mechanisms are more complex and varied ineukaryotes than in prokaryotes.

RNA POLYMERASE

Mediates transcription of RNA from a double-stranded DNA template

Does not require primer

Transcription is always 5’ --> 3’ direction, with each base addition using reaction RNAn + NTP --> RNAn+1 + PPi

Transcription inititation mediated by proteins that bind to specific promoter sequence elements. The promoter binding proteins recruit RNA polymerase to the initiation site.

RNA polymerases show evolutionary conservation between prokaryotes and eukaryotes, accounting for many similarities in transcription mechanism

But there are substantial differences in transcription initiation, termination, and post-transcriptional RNA processing between pro- and eukaryotes

PROKARYOTIC TRANSCRIPTION INITIATION

Prokaryotic promoters have elements near position -10 and -35 that bind

to the initiation factor.Promoter elements have been physically mapped by DNA footprinting.

Footprinting Technique

ELONGATION PROCEEDS IN A MOVING TRANSCRIPTION BUBBLE

TRANSCRIPTION - TRANSLATION COUPLING IN PROKARYOTES

Prokaryotic mRNA does not require processing nor cell compartment trafficking to become competent for translation.

Therefore, transcription and translation are coupled. In prokaryotes, gene regulation is almost exclusively at level of transcription initiation, and not later transcription or translation processes.

PROKARYOTIC TRANSCRIPTION TERMINATES BY RNA MECHANISM

(rho) - independenttranscription termination

signal motif

protein is recruited to RNA polymerase during elongationto allow transcription termination at other signal motifs

PROKARYOTIC RIBOSOMAL RNA IS GENERATED BYENDONUCLEASE PROCESSING OF A PRECURSOR TRANSCRIPT

THREE EUKARYOTIC RNA POLYMERASES SYNTHESIZEDIFFERENT TYPES OF RNA BY DIFFERENT INITIATION MECHANISMS

POL II INITIATION COORDINATED THROUGH A TATA-BOX PROMOTER

Transcription initiation factors werepainstakingly identified throughestablishment of cell-free in vitro transcription assays using TATA-box-containing DNA fragment, RNA Pol II, 32P-NTPs, and nuclear protein extracts.

Competent extracts were then subjectedto biochemical fractionations andreconstitutions.

EUKARYOTIC POST- AND CO-TRANSCRIPTIONAL RNA PROCESSING

Transcription elongation very similar in eukaryotes and prokaryotes: moving polymerase transcription bubble.

Little is known about eukaryotic transcription termination. 3’ ends of Pol II transcripts are generated by site-specific endonuclease cleavage and template-independent poly-adenylation.

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EUKARYOTIC POST- AND CO-TRANSCRIPTIONAL RNA PROCESSING

Nascent Pol II transcripts undergocapping of the 5’ end throughreaction with GTP in a 5’-5’orientation.

Capping protects RNA fromexonuclease degradation and provides a recognition site fortranslation initiation factors afterexport to the cytoplasm.

EUKARYOTIC POST- AND CO-TRANSCRIPTIONAL RNA PROCESSING

Nascent Pol II transcripts in most cases undergo splicing to remove intronic sequences and unite coding sequence exons.

POL II TRANSCRIPTION AND PROCESSING ARE COUPLED IN NUCLEUS

Capping, splicing, and 3’ cleavage/polyadenylation all occur while RNA is being transcribed.

EXON CHOICE IN SPLICING

Most frequently, splicing joins adjacent exons to assemble mature RNA.

For some genes, exons are “skipped” during splicing to generate alternative RNA products. Exon “choice” is tightly regulated, allowing generation of different proteins from same gene in different cells.

Different growth factor (FGF) receptors are synthesized due to selective use of exons 5, 6, or 7 into the mRNA. Epithelial cells use exon 6 (IIIb), but mesenchymal cells use exon 7 (IIIc). The alternative receptors bind to different FGFs.

IMPROPERLY SPLICED RNAs ARE DEGRADED IN THE CYTOPLASMBY NONSENSE-MEDIATED mRNA DECAY

Not all transcripts being made from a gene get spliced correctly. Many mis-spliced RNAs will contain nonsense codons and generate a highly truncated protein. Mis-splicing frequency can be enhanced by mutation.

WHY and HOW is the abnormal mRNA degraded???

IMPROPERLY SPLICED RNAs ARE DEGRADED IN THE CYTOPLASMBY NONSENSE-MEDIATED mRNA DECAY

As a messenger RNA is first translated, EJC proteins are removed as the ribosome passes by. Normally, translation termination will occur after all EJCs have been deactivated.

If there is premature termination, the translation release factor can interact with remaining EJC proteins, providing signal for degradation.

FOR SOME GENES, NONSENSE MEDIATED mRNA DECAY ISUSED TO AUTOREGULATE LEVEL OF GENE EXPRESSION

Pyrimidine tract binding(PTB) protein feeds backto induce mis-splicingof PTB pre-mRNA,thereby causing itscytoplasmic degradationby nonsense method.

EDITING OF PRECURSOR RNAs

MEASURING RATES OF A SPECIFIC RNA’S SYNTHESIS AND BREAKDOWN

Northern blotting or RNAse protection assay are used to measure amount of a specific RNA exists in a particular cell or tissue at particular time.

Differences in a gene’s RNA levels detected by these assays DO NOT reveal how why the RNA levels differ.

Nuclear run-on transcription assay measures ongoing rate of a gene’s transcription at a particular region along the gene. Therefore, this assay can determine whether differences in a genes total RNA abundance is the result of different rate of synthesis OR different degree of completion, as some genes have “transcription attenuation” sites.

Inhibition of new RNA synthesis with toxins (e.g. Actinomycin D) can be used to measure the stability (half-life) of a mature RNA species.