Naveen Sooknanan McGill Fall 2011
RNA Processing:
As we stated before, RNA polymerase II is the only one of its kinda to have a carboxyterminal
domain (CTD)
The CTD consists of the aa pattern YSPTSPS repeated
26 times in yeast and around 52 times in humans
o Is the CTD is removed in yeast, the organism dies, and the
same is assumed to happen for humans
The CTD is attached to Pol II by a linker peptide which is 28nm in
length
The CTD is a crucial part of Pol II because it is the source of two very
important phosphorylation events which essentially carry out RNA
processing of class II gene transcripts. These phosphorylation events are
carried out by the PNK activity of TFIIH
th
The first phosphorylation occurs on the 5 aa, a serine, on every repeat of the CTD. This
causes a capping enzyme to bind to the CTD and install a cap onto the 5 end of the pre-
mRNA as it is being transcribed
o This occurs after pol II has synthesized around 25 nucleotides
o This cap protects the new pre-mRNA from being digested by exonucleases
o After this phosphorylation occurs, pol II becomes much more active and enters an
elongation phase
The second phosphorylation occurs on the 2 aa of the sequence, also a serine, at every
repeat on the CTD
o This occurs during elongation and recruits the splicing machinery necessary for
splicing out noncoding regions of the pre-mRNA
Pol I and Pol III dont have CTDs, so their gene products do not undergo capping
o They do, however, undergo post transcriptional modification which will be
discussed later
Capping involves the addition of a 7 methylguanylate cap to the 5 end of the pre-mRNA as
soon as it exits the transcription complex
This ensures that the mRNA is not damaged by exonucleases and
happens so quickly because to the close proximity between the 5 end
of the mRNA and the capping enzyme on the CTD
o This 7 MG cap comes from a GTP substrate can creates an
odd 5 5 phosphodiester linkage which exonucleases cant
recognize
Animals and higher plants have a second security barrier in which the
second base is methylated on the 2 hydroxyl group
Vertebrates have a further security feature which methylates the 2
hydroxyl group of the third base
Along with evasion of exonuclease digestion, the capping also allows for the recognition
of various nuclear export proteins as well as translation factors in the cytoplasm
1Naveen Sooknanan McGill Fall 2011
The transition from pre-mRNA to mRNA is carried out by a mechanism called splicing. Unlike
bacterial genes, most eukaryotic genes have introns which must be spliced out before the mRNA
is translated.
While introns were initially considered to be junk DNA, it is found that they can actually
code for important regulatory functions as well as aid in alternative splicing events
Splicing was discovered by hybridization events in which a finals mRNA was annealed to its
corresponding gene
To the scientists surprise, the mRNA was actually much smaller than the gene
For example, in the hexon gene, DNA segments which coded for introns looped out of
the double stranded structure because they were spliced out of the final mRNA
Because of this size discrepancy, it was proved that genes pre-mRNA must undergo some
splicing event to reach a mature state
One important characteristic in splicing is the high level of conservation of intron borders. This
helps splicing machinery know where to carry out its splicing events to form mature mRNA
This is helpful for prediction of mRNA sequences
from a gene without conducting the actual
sequencing
An AG is highly conserved at 3 end the 5 exon, followed by a GU at the 5 end of the
intron
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