LECTURE 17:  RNA TRANSCRIPTION, PROCESSING, TURNOVER
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LECTURE 17: RNA TRANSCRIPTION, PROCESSING, TURNOVER. Levels of specific messenger RNAs can differ in different types of cells and at different times in the same cell. Control of RNA abundance can be at the level of transcription initiation, transcription elongation, processing or degradation.

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LECTURE 17: RNA TRANSCRIPTION, PROCESSING, TURNOVER

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Lecture 17 rna transcription processing turnover

LECTURE 17: RNA TRANSCRIPTION, PROCESSING, TURNOVER

Levels of specific messenger RNAs can differ in different types of cells

and 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 in

eukaryotes than in prokaryotes.


Lecture 17 rna transcription processing turnover

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


Lecture 17 rna transcription processing turnover

PROKARYOTIC TRANSCRIPTION INITIATION

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

to the s initiation factor.

Promoter elements have been physically mapped by DNA footprinting.

Footprinting Technique


Lecture 17 rna transcription processing turnover

ELONGATION PROCEEDS IN A MOVING TRANSCRIPTION BUBBLE


Lecture 17 rna transcription processing turnover

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.


Lecture 17 rna transcription processing turnover

PROKARYOTIC TRANSCRIPTION TERMINATES BY RNA MECHANISM

r (rho) - independent

transcription termination

signal motif

r protein is recruited to RNA polymerase during elongation

to allow transcription termination at other signal motifs


Lecture 17 rna transcription processing turnover

PROKARYOTIC RIBOSOMAL RNA IS GENERATED BY

ENDONUCLEASE PROCESSING OF A PRECURSOR TRANSCRIPT


Lecture 17 rna transcription processing turnover

THREE EUKARYOTIC RNA POLYMERASES SYNTHESIZE

DIFFERENT TYPES OF RNA BY DIFFERENT INITIATION MECHANISMS


Lecture 17 rna transcription processing turnover

POL II INITIATION COORDINATED THROUGH A TATA-BOX PROMOTER

Transcription initiation factors were

painstakingly identified through

establishment 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 subjected

to biochemical fractionations and

reconstitutions.


Lecture 17 rna transcription processing turnover

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.

s


Lecture 17 rna transcription processing turnover

EUKARYOTIC POST- AND CO-TRANSCRIPTIONAL RNA PROCESSING

Nascent Pol II transcripts undergo

capping of the 5’ end through

reaction with GTP in a 5’-5’

orientation.

Capping protects RNA from

exonuclease degradation and

provides a recognition site for

translation initiation factors after

export to the cytoplasm.


Lecture 17 rna transcription processing turnover

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.


Lecture 17 rna transcription processing turnover

POL II TRANSCRIPTION AND PROCESSING ARE COUPLED IN NUCLEUS

Capping, splicing, and 3’ cleavage/polyadenylation all occur while

RNA is being transcribed.


Lecture 17 rna transcription processing turnover

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.


Lecture 17 rna transcription processing turnover

IMPROPERLY SPLICED RNAs ARE DEGRADED IN THE CYTOPLASM

BY 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???


Lecture 17 rna transcription processing turnover

IMPROPERLY SPLICED RNAs ARE DEGRADED IN THE CYTOPLASM

BY 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.


Lecture 17 rna transcription processing turnover

FOR SOME GENES, NONSENSE MEDIATED mRNA DECAY IS

USED TO AUTOREGULATE LEVEL OF GENE EXPRESSION

Pyrimidine tract binding

(PTB) protein feeds back

to induce mis-splicing

of PTB pre-mRNA,

thereby causing its

cytoplasmic degradation

by nonsense method.


Lecture 17 rna transcription processing turnover

EDITING OF PRECURSOR RNAs


Lecture 17 rna transcription processing turnover

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.


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