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Lecture

BIOB10Y3 Lecture Notes - Rna Polymerase Ii, Transcription Preinitiation Complex, Tata Box


Department
Biological Sciences
Course Code
BIOB10Y3
Professor
Aarti Ashok

Page:
of 3
Transcription: tRNAs
50 different species of tRNA = transfer RNAs
o Encoded by repeated DNA sequences
o Internal promoter
o Transcribed by RNA polymerase III
o Lots of non-transcribed spacer sequences
o tRNA coding sequences in tandemly repeated arrangement
tRNAs match up the sequence of nucleotides with the correct amino acid to be inserted into the protein that is
encoded
o matches up to the mRNA
o each tRNA brings along an enzymatically attached amino acid
Transcription: mRNAs
mRNAs are synthesized from precursors that are found in the nucleus:
o radioactively label RNA nucleotides in cells (30min pulse) pulse experiment, no chase
o Lyse open the cell, extract the RNA and look to see where the radioactivity ended up
OR
o Wash out the radioactivity for 3 hours after the pulse = CHASE
o Then lyse cells, extract RNA and look to see where the radioactivity ended up
All eukaryotic mRNA precursors are synthesized by RNA polymerase II
o Enzyme has many different subunits
Requires the activity of various “general” transcription factors (GTFs)
o Called general b/c their binding is required for transcription to initiate at many different promoters
Polymerase II recognizes promoter regions located 5’ of the transcription unit
o b/w -24 and -32 is a specific promoter sequence known as the TATA box
o Polymerase II and the transcription factors assemble here = preinitiation complex
TATA box is bound by a protein known as the TATA-binding protein = TBP
TBP binding is required for RNA polymerase II to initiate transcription at the right place
TBP binds in the minor groove of DNA and significantly alters its conformation
o Bends the DNA
The assembly of the preinitiation complex occurs in a step-wise manner
TAFs = TBP association factors
TFIID (Transcription Factor 2 D) is a large complex of TBP and various TAFs
TFIIH assembles onto the complex once RNA Polymerase II is bound; 2 enzymatic functions:
1. Helicase unwinds DNA
2. Kinase phosphorylates Polymerase II
RNA Polymerase II assembled as part of the pre-initiation complex is not phosphorylated
o it becomes heavily phosphorylated upon initiation of transcription
o phosphorylated by TFIIH and other kinases
o on serine residues in its C-terminal domain (CTD)
*30 min PULSE only
-very large RNAs radioactive
-found to be in nucleus
-heterogeneous nuclear RNAs (hnRNAs)
*30 min PULSE + 3 hour CHASE
-hnRNAs not seen
-smaller RNAs
-found to be in cytoplasm
=hnRNAs processed into smaller RNAs that are exported to the cytoplasm = mRNAs
phosphorylation is thought to be the trigger that allows Polymerase II to escape the promoter and move down
the DNA template
Once Polymerase II begins transcription, most GTFs are either left behind at the promoter (TFIID) are simply
released from the complex (TFIIB)
o TFIID can then initiate assembly of a preinitiation complex with another RNA Pol II molecule
o additional rounds of transcription without the delay of assembly
Properties of mRNAs
they are found in the cytosol
they contain a sequence of nucleotides that will direct the order of amino acids in the proteins that they encode
they often contain untranslated regions (UTRs) that do not direct the incorporation of amino acids but play
important regulatory roles
o can be present on both 5’ and 3’ ends of the coding region
5’ terminii contain methylated guanosines = 5’ cap
3’ terminii contain 50-250 adenosines = poly (A) tail
They are transcribed from segments of DNA that are separated from one another along the template strand =
introns spliced out
Processing of mRNAs
There are 3 processing events that need to take place:
1. Addition of 5’ cap
2. Addition of a 3’ Poly(A) Tail
3. Splicing out of introns (fusing exons together)
5’ Capping
o building mRNA
o 5’ end has a triphosphate
o STEP 1
RNA Triphosphatase clips the terminal phosphate group, leaving a disphosphate
o STEP 2
Guanylyltransferase (which is the same enzyme as Triphosphatase, just a different active site)
then adds a GMP in an inverted orientation = 5’-5’ triphosphate bridge
o STEP 3
Position 7 of the guanine base is then methylated by RNA methyltranferase = 7-methyl-
guanosine cap
2’ position of the ribose to which the GMP was attached is also methylated
o FUNCTION of the 5Cap
Protection form digestion by exonucleases
Signal for export to the cytosol
Initiation of the mRNA translation
3’ Poly(A) Tails
o at the 3’ end of the mRNA, a different type of processing takes place
o add several A’s = polyadenylation poly(A) tail addition
o STEP 1
an endonuclease clips off part of the sequence on the 3’ end to create a new free 3’ end
o STEP 2
An enzyme known as poly(A) polymerase then adds an adenosine to the new 3’ end, w/o the
need for a DNA template
o 200-250 adenosines are added to the end of human mRNAs during processing
*NOT phosphorylated
-preinitiation complex on promoter
*phosphorylated
-initiates transcription
-elongation factors bound (SII, ELL)
-PTEFb is another kinase that phosphorylates CTD once initiation begins
o ~70-90 in yeast
o NOTE: not all eukaryotic mRNAs have poly(A) tails
o FUNCTION of the 3Poly(A) Tail
Protection from digestion by exonucleases
Aids in translation
Can be used to isolate mRNA from cell using oligo dT column
Splicing out of Introns (fusing exons together)
o Removing introns from mRNAs
Need to make breaks at the 5’ and 3’ ends of the intron and then join together the 2 exons that
are separated by that intron
o Splicing needs to be very precise
Addition or loss of even a single nucleotide could result in mistranslation =wrong protein
=wrong function
o How are the different intron/exon boundaries in thousands of mRNAs recognized for splicing?
Consensus sites
G/GU at 5’ end of intron, AG/G at 3’ end of intron
Preferred nucleotides near these sites site recognition
Polypyrimidine tract near the 3’ splice site
these are the essential core sequences needed for recognition by the splicing machinery but
these alone are not sufficient
additional sequences known as exonic splicing enhancers (ESEs) situated in exons are
also required for recognition to lead to splicing
Properties of mRNAs
They are transcribed from segments of DNA that are separated form one another along the template strand
o The majority of eukaryotic genes contain intervening sequences
o cDNA (complementary DNA) = mRNA sequence that has been reverse transcribed to a DNA sequence
using special enzymes called reverse transcriptases DNA that is the exact sequence of mRNA
o restriction enzyme cleavage results in vastly different sized fragments; genomic DNA contains
intervening sequences that are removed in the cDNA (mRNA)
split genes are the rule, not the exception in eukaryotes
all genes, including those that encode tRNAs, rRNAs, and mRNAs all contain introns (intervening sequences) and
exons (coding regions)
Processing of mRNAs
larger, primary RNA is made first from the DNA sequence, then processed to mature mRNA [hnRNAs mRNAs]
RNA-DNA hybrids: mRNA sequences pair up with the complementary sequences in the DNA
o The pre-RNA sequence of the globin mRNA and the DNA (gene sequence)
o Shows that the pre-RNA sequence is exactly matched up to the DNA
o So, RNA first made containing all intron and exon sequences of split genes (hnRNAs)
Then, if we take the mature mRNA of the globin gene and the DNA (gene sequence)
o It shows that the mature mRNA sequence lines up in most areas but it lacks complementarity with some
regions of the DNA
o These regions loop out = introns
o So, mature mRNA does not contain introns and is instead made up of exons that are fused together =
introns are “spliced” out