Lecture 8

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25 Mar 2012
Lecture 8: Transcription 2
1. Structure of a gene
2. RNA polymerases
3. Transcription of protein coding genes
4. RNA processing
An expanded repertoire of RNAs to generate
oIn eukaryotes we have RNA species which extend well beyond those normally
required for simply production of polypeptides
oTo make all these different RNAs you also need different RNA polymerases
oIn bacterial system we focus on 1 single RNA polymerase but in eukaryotic
system we focus on multiple RNA polymerases and they generate different
types of RNA
osnRNAs and snoRNAs have particular function involved in helping to process
other RNAs forming secondary structures, binding to those RNAs, facilitating
the splicing and reorganization of mRNAs or rRNAs
oscaRNAs used to modify help splice, reorganize snRNAs and snoRNAs which
are then used to help splice the preRNAs
omiRNAs and siRNAs are important in regulating gene expression. They are
being used by the cell at particular stages of post transcription or translation
regulation by blocking either the translation of specific RNAs or by binding to
existing RNAs through complimentary base pairing
A number of eukaryotic RNA polymerases required
oRNA polymerase 1 is involved in primarily in ribosomal RNA gene production
oRNA polymerase 3 is involved in tRNAs
oEach of these RNAPs is a multi-subunit protein
oEach is responsible for transcription of different sets of RNAs
Subunits of eukaryotic RNA polymerases
oThe 5 subunit complexes (beta, beta’, alpha1, alpha2, omega) are all you need
to produce bacterial functional RNA polymerase
oVariety of other subunit complexes for eukaryotic RNA polymerases
oC terminal domain (CTD) is a little tail that extends out of the core of subunit 1
in RNA polymerase 2
oSome subunits resemble the subunits of bacterial RNAP’s
Eukaryotic vs. bacterial RNA polymerases
oEukaryotic RNA polymerases require proteins to help position them at the
promoter called transcription factors
oThese factors fulfill a similar role to the sigma subunit of the bacterial RNA
oThe reason for these factors is because eukaryotic RNA polymerases need to
deal with chromosomal structures
Gene structure
oProkaryotic mRNA use polycistronic message to produce 3 proteins in this case
oEukaryotic mRNA don’t encode more than a single gene within that mRNA but
they also require significant amount of processing
oIt involves things such as, addition of cap structures, Poly-A tails, etc.
Transcription of protein-coding genes
oInitiation of transcription
oRNA processing
oPromoters are the consensus sequences that are found in the genomic DNA
which are recognized by transcription factors and they initiate the process
such that the RNA polymerase can wind up in the correct spot
oTF2B: transcription factor RNA polymerase 2 B
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oPositioning of these promoter elements is positioned relative to the
transcription starting point so in fact in eukaryotic transcriptional activities,
promoters can be part of the RNA that is being transcribed
oFor instance, DPE element is going to be part of the primary transcript when it
is made but it also acts as a promoter element as it’s positioned on the DNA
oSame for the rest of the elements, they are 5’ transcriptional starting point, so
they wouldn’t be transcribed
oINR is almost at the transcription starting point
oTATA box is most often seen and it is recognized by TATA binding protein which
is in fact, a subunit of transcription factor 2D (TF2D)
oTATA element causes conformation shifting of the helix it bends the helix
quite significantly
oThe lump in that double helix configuration than acts like a geographical signal
for the rest of the transcription factors to now start to associate in that region
oNot all promoters for genes contain all these elements but these elements are
most often found in eukaryotic genes
Steps in the initiation of transcription
oInvolves the aggregations of general transcription factors to the sites on the
DNA where it can now mobilize the RNA polymerase
oTATA box is an aligomer (combinations of different protein complex)
oTBP is the TATA binding protein which is part of the TF2DD complex
oIt first involves the binding of TBP along with the rest of the TF2D polypeptide
to the TATA binding
oIt mobilizes the binding of the TF2B protein complex to the region on adjacent
to TATA box
oOther protein factors get associated with that location and now start to provide
a region for a RNA polymerase to coordinately the bind
oTF2H is a large polypeptide that encodes a number of specific functions. It also
encodes a helicase so it’s going to be involved in unwinding of DNA. It also
contains a number of kinase domains which are going to be involved in
phospholating RNA polymerase as it goes into elongation phase
oAll together, this is called transcription initiation complex
oC-terminal domain (CTD) is a protein extension that is found specifically in RNA
polymerase 2 and it’s an element of RNA polymerase that is highly
phospholated regulated and actually plays an coordinating role in the activity
of this whole complex
oOne of the key factors involved in the initiation of transcription is this
phosphorelation of C-terminal domain and it changes the configuration of RNA
polymerase on DNA strand
oTATA binding protein, which is a subunit of TF2D, binds to the TATA box,
bending and distorting the DNA
oThis attracts transcription factors, which help to orient and bind RNAP2 to the
oHelicase activity of TF2H uses ATP to open up the DNA strand
oTF2H also phosphorelated the C-terminal tail of RNAP2, activating it so that
transcription can begin
RNA polymerase 2 C-terminal tail
oIt has a series of repetitive amino acids motifs
oIt is going to be highly regulated tether, almost like a chain where
phosphorelation events now act as attractants for other polypeptides that are
going to be required
Additional factors required for transcription initiation
oThere are lots of other proteins which are involved in transcription complex
which include things such as, histone modifying enzyme, chromatin
remodeling complexes, etc. that help the RNA polymerase navigate through
nucleosome structures, loosen the DNA from the histones and so on
oEukaryotic gene expression involves many more complicated steps associated
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