BIOL 200 Lecture Notes - Zinc Finger, Hybridization Probe, Electrophoresis

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6 Apr 2012
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Naveen Sooknanan McGill Fall 2011
1
Eukaryotic Transcription:
Although the main process of eukaryotic transcription is very similar to that of prokaryotes, the
actual factors involved make the mechanism much more complex
Initiation of translation is particularly complex in eukaryotes
Requirement of an RNA polymerase, nucleotides, complement bonding, all still applies
to eukaryotes
Eukaryotic transcription is limited to two or three organelles
Transcription in the mitochondria and plastids (in plants and algae) have a process
mechanically similar to prokaryotes
Transcription in the nucleus is by far the most important and complex
There are three eukaryotic RNA polymerases, each differing in gene products and in their
sensitivity to the mushroom poison mannitin
RNA polymerase I produces mainly rRNA precursors such as 28S, 5.8S and 18S
o Pol I is not sensitive to mannitin
RNA polymerase II is the one we will focus most on, and is responsible for all protein
coding genes (mRNA) as well as factors involved in splicing, and other non-coding
functional RNA (which we don’t understand)
o Pol II is highly sensitive to mannitin
o Pol II is the only ATP dependant RNA polymerase
RNA polymerase III is responsible for the transcription of tRNAs, a splicing factor
called U6, small stable RNAs and rRNAs produced outside the nucleus
o Pol III is mildly sensitive to mannitin
All three polymerases are multi-subunit complexes consisting of more than 10 subunits. They are
all very similar to prokaryotic RNA polymerase, showing little evolutionary change.
These RNA polymerase molecules have no idea what needs to be transcribed and what
doesn’t, so they need the help of some transcription factors which will direct them to the
correct genes
Pol II is the only RNA polymerase which has a carboxyterminal
domain (CTD)
o The CTD is a repeat of 7 amino acids ~52 times in humans and
26 times in yeast
o It is very useful in downstream reactions, as we will see later on
These subunits are all very crucial in the functionality of the RNA
polymerase. Without one, the organism would die
Upstream of the protein coding region of DNA is a region rich in As and
Ts. This region is known as the TATA box and is very highly conserved.
The TATA box, 8 nucleotides long, is normally located between -35
and -25 on the gene and acts a promoter to mark the beginning of
the transcription process
Some eukaryotes, which are not well understood, are mediated by an
initiator sequence which is not well defined
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Naveen Sooknanan McGill Fall 2011
2
The TATA box binding protein (TBP) does exactly what its name says; binds to the TATA box
TBP binds to the minor groove of the DNA using its conserved CTD and causes a radical
bending of the DNA structure where the TBP sits down
This protein has been purified and structurally analyzed, and, strangely enough,
eukaryotes without a TAT box still need the TBP in order to initiate transcription
The binding of TBP to the TATA box then calls in an armada ot transcription factors (TFs) as
well as Pol II to initiate transcription and get it ready for the elongation phase
Once the TBP binds, TFIIB recognizes the bent structure and binds to the TBP forming a
complex
Pol II, which is always associated with TFIIF then joins the complex
TFIIE then joins this complex
The complex is then recognized by TFIIH which is a very important enzyme in this
process
o TFIIH has 2 helicase functions as well as one PNK function
o Once TFIIH binds, the pre-initiation complex is formed
TFIIH, through its protein kinase activity, phosphorylates the CTD of Pol II which
activates it
o The CTD becomes hyperphosphorylated, meaning multiple phosphates are added
on
Through the helicase activity of TFIIH, the DNA is unwound to form a transcription
bubble
Through the phosphorylation of Pol II, all the general TFs leave except the TBP
Once the CTD of Pol II is hydrolyzed, system switched from initiation to elongation
TFIIH is an energy dependant enzyme which uses ATP both in its PNK activity as well as its
helicase activity (melting the base pairs)
One of the helicase activities of DNA helicase is a proofreading ability. This is useful for
picking up mutations in the DNA as it scans along
o One important mutation is the formation of thymine-thymine dimers which can
happen in the presence of UV light
Patients with a cancer predisposing disease called Xeroderma pigmentosum cannot enter
sunlight because their nucleotide excision repair (NER) system cannot properly repair
these thymine-thymine dimers and transcription of all proteins becomes erroneous
o These patients are much more likely to develop skin cancer
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