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Lecture 8

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University of Toronto St. George
Jennifer Harris

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 structure. 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 transcribing RNA polymerase is the transcribing protein Slide 3 DNA helix 5’ new DNA DNA/RNA - 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  Termination - 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 shape. 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  Promoter - 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 st RNA polymerase – from the little fragment that it needs to bind 1 , 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 is not so important; can be anything. The size of the gap has constraints on it – always 15-19 nucleotides. If you had these 2 sets of consensus sequence separated by shorter amounts, even if you have these exact nucleotides, if they’re separated by only 1/2 nucleotides, sigma subunit is not likely to bind. There is some variation in the size of the gap but on average, 17 is optimal size. - This is what is meant by consensus sequence bound by sigma subunit – incorporates this whole concept. The percentage of binding if you have a T or an A or a G in a particular place as a promoter region. Slide 12 1. Description about what types of promoter sequences the sigma factors are likely to bind & they represent percent averages across alignments of nucleotides. 2. Promoter strength varies – can have promoter sequences that are very optimal in binding sigma units which would result in huge amounts of transcription of that particular gene. For weaker promoters – contain alternative nucleotides at some of those consensus sequences & those alternatives reduce strength of promoter – sigma subunit doesn’t bind as well – not transcribing as many mRNAs. 3. The +1 (in the numbering diagram showing position of consensus sequences). 4. Orientation – If they were not asymmetric sigma subunits would bind on either strand – not generally what is desired/required in cell. Important that any particular promoters controls the genes downstream of it.
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