Eukaryotic gene regulation - Professor Latchman Flashcards
What is the branch point?
The point where the G from the GU sequence binds to the A in the intron to form the lariot is called the branch point. The A that forms the 2’ carbon bond with the G is called the branch point.
Describe in full detail the process of RNA splicing
U1 particle recognises the 5’ splice site, it recognises the highly conserved GU sequence as well as the less well conserved polypyrimidine tract. U1 binds to the GU sequence.
U2 particle recognises the A branch point and binds to it, again it recognises the highly conserved A at the branch point but it also recognises the less well conserved sequences around this point, these sequences can change from intron to intron. U2 particles binds to the A branch point.
U1 is displaced by the U4/U6 particle which recognised the U1 bound to the GC sequence. This releases the U1 and the U4 particles from the complex, leaving just U6.
U5 binds to a specific region upstream of the exon 1
U2 and U6 now interact with each other, this brings the GU and the A branch point close together so that a reaction can occur. The phosphodiester bond between the 5’ phosphate of the intron and the 3’ oxygen of the upstream exon is exchanged for a phosphodiester bond between the 5’ phosphate of the intron and the 2’ oxygen of the A branch point. The lariot structure now forms. U5 now moves into the intron. After the U2 particle has bound to the A branch point this targets the next AG in the sequence downstream of the branch point to be the 3’ splice site. The phosphodiester bond between the 5’ phosphate of the downstream exon and the 3’ oxygen of the intron is exchanged for a phosphodiester bond between the 5’ phosphate of the downstream exon and the free 3’ oxygen of the upstream exon. This joins the two exons together and releases the intron in a lariot structure.
What is the intermediate structure of RNA splicing?
The lariot structured intron is attached to exon 2.
What is a spliceosome?
A complex structure where splicing takes place, it contains both proteins and RNA molecules known as URNAs. Each URNA and its associated protein (sn proteins) bind to different parts of the RNA that is being spliced and catalyse the process. Also the fact that the splicing occurs in the spliceosome, this prevents exon 1 from diffusing away once it has been cut.
What is the role of RNA helicase Prp22?
This RNA helicase binds to the downstream exon after splicing and moves along the spliced RNA in a 3’ to 5’ direction, releasing it from the spliceosome.
What is the role of RNA polymerase I?
To transcribe the rRNA genes. It does this by transcribing lots of repeating precursor DNA called 45S, and then the resulting mRNA is cut up into the smaller rRNA that the cell needs, 28S, 18S and 5.8S. There are several hundred of the rRNA 45S precursor genes in a repeated unit.
Describe how RNA polymerase I transcription initiates
A protein factor called UBF recognises a conserved sequence at around -50 on the 45S gene. UBF acts as the upstream binding factor.
This binding results in another factor called SL1 being recruited, SL1 doesn’t recognise a specific sequence on the DNA it just recognises UBF which is bound to a specific sequence on the DNA.
Only when SL1 and UBF are bound can the RNA polymerase I recognise the complex and bind as well.
Why is the transcription initiation of RNA polymerase I less regulated then that of RNA polymerase II?
RNA polymerase I transcribes rRNAs compared to proteins. All cells need rRNAs, not all cells need all types of proteins, therefore the polymerase that transcribes the proteins needs to be more highly regulated in order to turn it off when it is not needed.
Describe the synthesis of tetrahyene rRNAs
It is a very similar process to the synthesis of eukaryotic rRNAs, it involves a big precursor molecule that is cut up into the smaller rRNAs. The precursor DNA has an intron in its 3’ end, this intron has to be spliced out after it has been converted into RNA via RNA splicing. The RNA splicing occurs after the precursor RNA has been cut into its three parts. This is a very simple form of splicing, all that is needed is an inorganic salt solution containing Mg or Ca. If the concentration of the solution is correct then the intron will remove itself, in this system RNA introns seem to be able to fold up and splice themselves, this is called self-splicing.
What does RNA polymerase III transcribe?
RNA polymerase III transcribes the genes for the 5S ribosomal RNA and the tRNAs. RNA polymerase III also transcribes snRNAs such as U6, all of the others are transcribed by RNA polymerase II.
Where is the promoter for the precursor gene that RNA polymerase III transcribes in order to make 5S rRNA?
In the centre of the RNA coding sequence, between +40 and +80. Researchers found this by cutting away pieces of the gene and seeing what effects this had on transcription, only when they cut the middle of the gene did they see an effect.
Describe the mechanism for the transcription initiation by RNA polymerase III at the 5S rRNA gene promoter
A protein called TFIIIA recognises the internal promoter at around +50 and binds to it.
After this binding TFIIIC recognises that TFIIIA has bound to the specific sequence and now TFIIIC binds to TFIIIA. TFIIIB now binds closer to the +1 site and it is when all three of these proteins are in position that TFIIIB recruits RNA polymerase III.
Do RNA polymerases recognise specific sequences on the DNA?
NO! No RNA polymerase ever binds to a sequence on the DNA, RNA polymerases bind to proteins that have bound to specific sequences.
How does the 5S rRNA transcribed by RNA polymerase III have an inbuilt control mechanism?
As the promoter is in the middle of the rRNA gene then it has two roles, it has to form part of the functional rRNA structure but it also has to initiate transcription by binding to TFIIIA.
TFIIIA can bind to both the DNA and the rRNA, therefore if too much rRNA is being made then the TFIIIA will be more likely to bind to the lots of rRNA as opposed to the DNA and this will reduce the amount of transcription that occurs. However, if there is not enough rRNA being made then it is more likely that the TFIIIA will bind to the DNA and initiate transcription so that more is made.
Describe SDS page as a method of analysing proteins in a cell type
The proteins are treated with a detergent (SDS) to remove their charges and then they are run on a gel electrophoresis to separate them by size. So we can compare the proteins inside different cell types by their size. This is however, a very low resolution way of studying proteins in a cell as it is very easy to have two proteins that have the same size and you would not be able to tell them apart. A way to get around this is to do 2D SDS page electrophoresis.
