Lecture 32: RNA Synthesis Flashcards
4 types of RNA from DNA templates
mRNA
rRNA (ribosomal)
tRNA
small nuclear RNAs
Bactreial RNA polymerases
bind to specific DNA Promoter Sequences (on 5’ end)
initiatin of synth starts with open complex formation and copy the template strand
3 RNA polymerases in eukaryotes
Pol 1 (synth rRNA) Pol 2 (synth mRNA) Pol 3 (synth tRNA, rRNA)
mRNA
directs messages and sequence of amino acids in protein
rRNA
large, part of ribosome
catalytic RNA
tRNA
link between mRNA and amino acid
base pairing of what?
tRNA and mRNA
snRNA
regulatory functions
Transcription
DNA dependent RNA synth
aka Gene Expression
difference between transcirption in eukaryotes vs prokaryotes
Eu: transcribe DNA in nucleus, translate mRNA in cytoplasm. processesd by RNA splicing
Pro: transcription and translation at same time
RNA splicing is to…
remove non-coding sequences (introns)
if templates strand DNA is… then RNA is…
G
A
T
C
U
A
if CODING strand DNA is G… RNA is…
G
RNA polymerase in prokaryotes
need to accesss template starnd of DNA to guide RNA synth
DOES NOT require pre-existing primer
directoion of RNA synth
5’ to 3’
base pairing in bacteria
leads to temp DNA-RNA hybrid at TRANSCRIPTION bubble
bacteria template strand
RNA will be complimentary to this strand
bacteria coding strand
has same sequence of RNA being created
complementary to template
Transcirption bubble…
moves base pairs reseals as complex moves opens so that complementary pairing can occur
DNA hybrid is..
pretty short
becomes single stranded as it exits
enzymology of RNA synth
like DNA synth
nucleophilic attack on 3’ OH group on alpha phosphate
generation of new phosphodiester bond with basepairing
RNA polymerase proofreading?
not clasic proofreading activity
but does “back-up” and resynthesize strand when mismatch is made
importance of magnesium ions in RNA pol
positive charges interact with negative charges of phosphate
stabilization
positioned by aspartate residues
Transcription generates _____ mRNA molecules
MANY!
each gene produces 1000 to 100000 transcripts
error rate is about 1 error per 10,000 nucleotides
(VERY similar to DNA polymerization error rate)
RNA pol does not have proofreading activity, and there is no RNA repair mechanism. Why is an error rate of 10^-4 nucleotides acceptable?
errors won’t persist because proteins made will only cause a temporary problem
odds of one being bad is lower???
winding problem
as transcription bubble moves along the DNA double helix, it creates stress and induces super coils on both sides of bubble
causes Torsional stress
what relives torsional stress?
topoisomerase
True or false? RNA polymerase transcribes only one DNA strand
FALSE
it transcribes both
CODING AND TEMPLATE STRANDS AREN’T ABSOLUTE
regions may differ
there is not much overlap so that proteins aren’t coded in both template and complementary strand
Where does RNA synth begin?
at promoter sequence
promoter sequences in bacterial genes
evolutionary conserved DNA sequences that are
-35 and -10 regions relative to +1 RNA start site
spacers between these 3 regions
when does the sigma factor fall off?
when the polymerase moves away from the promoter sequence (the minus regions)
- 10 and -35 regions recognized by…
sigma factor
directs -35 and -10 regions to form initial closed complex
Bacterial RNA pol: binding
RNA pol binds to promoter region as complex containing sigma factor
closed complex formed
complex binding leads to open complex
transcription starts and sigma factor falls off (sigma used again)
DNA footprinting
to find where RNA pol binds to DNA sequence
lable the DNAs, split into 2 grouos, 1 w/ protein of interest added, one w/0
add DNAse1 (enough to cut DNA once)
then we end up with DNA cuts of different lenghts
get cuts at every position
IF PROTEIN IS PRESENT, cutting can’t happen b/c DNA is protected by binding of RNA Pol
DNA suspected radilabled, incubate w/ or w/o RNA pol
DNA footprinting… what happens?
you end up with fragments that are cut at different sites
you can see where the RNA pol binds by running on a gel
3 conditions for footprinting
1) DNA must be labled on only 1 end of fragment (reference point)
2) DNase 1 digestion must be incomplete to generate the different fragment lengths
3) RNA pol must bind specifically to single sequences w/in fragment
DNA footprinting: Blank regions
is where it is bound by RNA pol, so protected from cleavage (bands only show up where there was cleavage)
(if we had no protein, cutting could happen anywhere)
blank regions are where “footprinting” is occuring
STUDY SLIDE 13
STUDY SLIDE 13
template strand is…
SEE SLIDE 14 TOO
complementary to growing RNA strand
so T would encode A, A would encode U… etc.
the complimentary coding strand is the same as the RNA strand
Eukarotic Polymerases: RNA pol 1
makes ribosomal RNA
promoters: INR sequence and UPE (upstream promoter element) element
RNA Pol 2
makes messenger RNA
promoters: INR sequence and TATA box (or DPE sequence)
RNA Pol 3
makes tRNA and one of rRNAs
promoters: blocks
A block and C block (for rRNA)
A block and B block (for tRNA)
how does an RNA pol know which RNA it will be making?
recognition of promoters
also, the pols work differently
alpha amanitin
inhibits Pol 2 high concs inhibit Pol 3 doesn't inhibit Pol 1 Why is this important? used to find differences between the pols
TATA binding Protein (TBP)
directs RNA Pol II compex to a T-A-T-A sequence in the promoter region of protein coding genes
TATA sequence positions the RNA Pol II complex w/in the gene promoter
DNA piece is really bent. TATA is easier to bend and open up, makes it easier to form open complex
RNA Pol 2 and promoter sequence
needs to recognize promoter sequence
binds to TATA and D. Add factors and RNA Pol 2. HUGE complex
once complex is made at promoter, transcription can start
then a bunch of factors fall off. D, TATA, 2F, and Pol 2 are all thats left
Pol 2 in Yeast
has 12 subunits
RNA pol complex unwinds DNA helix
polymerizes nascent mRNA transcript
lots of other stuff happens too
What is the bacterial equivalent of the TATA binding protein (TBP)
Sigma factor!
both help to position polymerase at right place to start transcription
not related at all structurally
