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LECTURE 18: 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.
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 s 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
r (rho) - independent
transcription termination
signal motif
r protein is recruited to RNA polymerase during elongation
to allow transcription termination at other signal motifs
PROKARYOTIC RIBOSOMAL RNA IS GENERATED BY
ENDONUCLEASE PROCESSING OF A PRECURSOR TRANSCRIPT
THREE EUKARYOTIC RNA POLYMERASES SYNTHESIZE
DIFFERENT TYPES OF RNA BY DIFFERENT INITIATION MECHANISMS
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-boxcontaining DNA fragment, RNA Pol II,
32P-NTPs, and nuclear protein extracts.
Competent extracts were then subjected
to biochemical fractionations and
reconstitutions.
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
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.
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 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???
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.
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.
EDITING OF PRECURSOR RNAs
MEASURING RATES OF A SPECIFIC RNA’S SYNTHESIS AND BREAKDOWN
Northern blotting, RNAse protection assay, or RT-PCR 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 what causes 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. Or
drive transcription of specific RNA ectopically from strong promoter
and measure steady-state abundance.
EXAMPLES OF RNA CONTROL MECHANISMS
Growth factors and hormones control rates of transcription initiation
at specific genes (nuclear run-on assay)
C-myc proto-oncogene expression level is controlled by transcription
attenuation. Some cancer cells show failure to induce c-myc
transcription attenuation
Heat shock controls levels of Hsp70 RNA by suppressing a rapid
RNA degradation mechanism