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Lecture 8

BIO120H1 Lecture Notes - Lecture 8: Stem-Loop, Sigma Factor, Consensus Sequence

Course Code
Jennifer Harris

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Lecture 8: Prokaryotic Transcription
Lecture Outline:
1) Prokaryotic transcription
2) Diversity of RNA structure & function
Readings: Alberts, Ch 6, pp. 329-339 Ch 8, pp. 573-575
Which statement is FALSE concerning ClustalW?
a) It is program which aligns multiple sequences to each other.
b) It requires input sequences to be FASTA format.
c) It will align any sequences together, even those chosen at random from sequence databases.
d) It uses algorithm which inserts gaps to maximize alignment score.
e) None of the above.
- a is what the program is actually for so that is true
- b is true b/c it’s the file format it uses, not mentioned in the lecture but now we know (like NEXUS for the
phylogenic program)
- c is true because it is an algorithm and doesn’t differentiate between real and fake sequences, just does what
its programmed to do
- d is true because gap insertion for better fit is one of its functions
- How the DNA information is used for the synthesis of RNA
- Prokaryote transcription
Slide 1 Transcription (creating RNA)
- Transcription is 1 of the cellular processes. Already talked about
process of DNA replication & how it replicates itself in a cell.
- In a cell, the DNA gets transcribed into RNA RNA is essentially an
intermediate between DNA & protein. Single stranded mRNA is
translated to a protein sequence which then folds into its tertiary
Slide 2 - Electron micrograph showing transcription in the cell.
- All the feathers/hairs coming off of DNA molecule are transcripts being
generated by RNA polymerases each 1 of dots are RNA polymerases
with emerging transcript coming off of it. These dots at the end are the
beginning of ribosome assembly. This is in prokaryotes where this can all
happen at once.
RNA polymerases are all along the DNA
We have all of the info for all the different RNAs that we can possibly
make but in any individual cell, we only transcribe a subset of mRNA
Enzyme making the RNA needs to seek out the right place to start
RNA polymerase is the transcribing protein
Slide 3 DNA helix
5’ new DNA
- RNA polymerase is synthesizing a new transcript from 5’ to 3’ means
that template strand from which it’s being synthesized is going from 5’ to
3’ – template strand must be dissociated from the other strand of DNA so
it can be available for base-pairing for the nascent RNA.
- DNA helix is moving off as this is being read ribonucleoside
triphosphates enter through the tunnel within the RNA polymerase itself
& the right nucleotides are the ones which base-pair with template & get
incorporated into the RNA (nascent mRNA chain).
- In this binding pocket of RNA polymerase, you have an RNA-DNA
hybrid in the chamber template strand being DNA & actively
transcribed RNA. Hybrid also needs to be separated at the end parent
DNA strand goes one way, the newly synthesized RNA goes through
RNA exit channel.
- The whole complex moving towards 3’ direction.
Slide 4 - RNA polymerase is actually very well designed for its function. If you
look at the 3D structure, there is very little room in the RNA polymerase
very tightly bound, surrounding both the DNA as well as the short
fragment of the DNA-RNA hybrid.
- There is very little room happening in there, the DNA is coiled around
the structure very tightly.
Slide 5 - The ancient protein domain (double psi barrel) gave rise to all RNA
polymerases (whether prokaryotes or eucaryotes). What is thought to
have happened is that this ancient double psi barrel dimerized (2 copies
of it 2 of same molecule gets connected together) & subsequently there
was the acquisition of some important AAs for functioning of RNA
polymerase such as lysine residues which are thought to be critical for
positioning of DNA template within RNA polymerase. Acquisition of
some aspartic acids which are thought to help stabilize & kelate Mg at the
active site.
- In addition to the basic structure which are 2 double side barrels, there
are also large loops which loop out – those were acquired later as well.
- RNA polymerase has been highly conserved in evol’n.
Slide 6 RNA polymerase
Sigma factor
Unwinds DNA
- Particular subunit of polymerase called sigma factor analogous to
eucaryotic transcription factors (much more complicated than prokaryotic
ones). Function of this sigma factor is to recognize sites of initiation of
transcription & to help position the RNA polymerase in the right place.
Sigma factor recognizes specific promoter sequences 2 consensus
sequences which are separated in the DNA strand & binds to it & helps
load polymerase onto that site of transcription initiation.
- Get the unwinding of the DNA & the allowance for RNA polymerase to
start transcription.
Slide 7 Initial RNA
RNA elongation
- Once you have that happening, get formation of initial small fragment
of the RNA. RNA polymerase currently in the open phase not a very
efficient phase of transcription initial fragment formed very slowly
seems RNA polymerase is testing and rejecting lots of nucleotides.
- Eventually, long enough fragment formed that allows RNA polymerase
to go into closed phase in which it can elongate RNA much more rapidly.
This is phase where most of RNA is synthesized.
- Then near the end of the transcript, you get this loop formation which is
a termination signal which induces the flap of the RNA polymerase to
open, releasing the completed mRNA transcript. Termination loop is a
secondary structure formed in the mRNA itself essential for
transcription to terminate & newly synthesized mRNA to be released.
Slide 8 1. Core (domain) + sigma factor sigma factor required for recognition
of promoter region for initiation of mRNA synthesis.
2. No; polymerase changes shape: RNA polymerase doesn’t require ATP
unlike DNA polymerase. RNA polymerase itself actually changes its own
3. Complementary need to have the correct base pairs forming & RNA
& DNA can form the appropriate base pairs, just like in DNA replication.
4. 5’ 3’ (direction like in all other polymerases).
- This lack of requirement for ATP is b/c it’s the movement of the RNA
polymerase that uses the energy contained in forming phosphodiester
bonds which is actually driving polymerase forward so opening the helix
is actually energetically favourable.
Slide 9 5. Inefficient (due to it being in the open configuration)
6. Processive Once it’s in the closed configuration it’s actually tightly
clamped around the DNA template which means that it does not tend to
fall off & that makes it very processive. Remember: DNA polymerase
had slightly different mechanisms to make it processive required a
sliding clamp to hold it onto the template. With RNA polymerase there is
actually change in conformation of the RNA polymerase itself which
makes it processive.
7. Hairpin – loops that forms on end of the mRNA.
A-T rich also there has to be this dissociation b/c the mRNA and the
DNA template so those the sequences at the end tend to be more A-T rich
b/c there are only 2-H bonds and they are easier to dissociate.
8. Hairpin opens flap the hairpin loop helps to pry open the flap that’s
actually holding RNA transcript within the RNA polymerase.
Slide 10 RNA polymerase
- Synthesis of RNA is always 5’ to 3’ but it can be actually on either
strand & which strand it’s actually reading as template depends on which
strand the RNA polymerase is loaded.
- This is example of a piece of bacterial genome – you have genes that go
in either direction can be oriented randomly in either direction &
sometimes can even have overlapping reading frames. (Ex: Genes C will
be overlapping with transcript for Gene D in other direction it’s
relatively rare b/c that imposes unique constraints on evol’n of sequences
b/c they’re coding for more than 1 thing at that point).
- What actually determines what strand is going to be transcribed?
Determined by orientation of promoter sequences it is promoter
sequences are the 1s that the sigma subunits that initially recognize &
bind to the DNA.
- Ultimately, the promoter sequence determines the orientation b/c it
determines the orientation of RNA polymerase from the little fragment
that it needs to bind 1st, it will synthesize in respective direction
depending on whichever strand its attached to.
Slide 11 - Promoter sequences are sequences that are recognized by the sigma
subunits. Is there is single, consensus sequence that these sigma subunits
bind to? No - they tend to bind with higher affinity to certain types of
sequences but they can potentially bind to a wide range of them.
- Graph is telling us that orienting of position of the nucleotide with
regard to first codon position. At codon position upstream of start site
tend to have T (represented by yellow) in almost 75% of the consensus
sequences bound by sigma subunit, would have T in this position. The
other 3 nucleotides all occur with very low frequency here.
- Even though there is no single sequence that sigma subunit will bind
there are sequences that are more likely to bind than others – so sequence
that has TTGACA will have high probability of binding than for a
sequence like AATGAT.
- The important nucleotides are actually separated by a gap stuff in b/w