Controlling gene expression Flashcards
Isoforms - what are they? how frequent are they in the human and drosophila genome?
Relation to splice donor sequences?
There are different forms of a protein made from the same gene. They arise due to alternative splicing of genes. 75% of the human genome produces isoforms and 40% of drosophila genes.
They occur because splice donor sequences are only 2 bases long so very common. The secondary structure of the protein can often impact the splice site.
Alternative splicing methods
Mutually exclusive axons - 2 or more exons spliced together
Internal splice site - area WITHIN AN EXON spliced
Optional exon - exon removed
Optional intron - intron removed
REGULATING ALTERNATIVE SPLICING - SEX DETERMINATION IN THE FRUIT FLY
Sex determination in the drosophila is determined by 3 genes - Sxl, Dsx, Tra.
Dsx - the male repressor protein (REPRESSES GENES THAT CAUSE FEMALE DEVDLOPMENT.)
In males Dsx is expressed and Sxl transcripts are spliced out, inactivating the protein.
Females have a small amount of Sxl - which acts as an alternative promoter and represses splicing, and its own positive feedback.
Sxl binds to Tra transcripts and this causes production of a female-specific Dsx isoform. This REPRESSES MALE GENE DEVELOPMENT
Regulating mRNA polyadenylation - example is B lymphocytes?
B lymphocytes can produce antigens and either express them on their cell surface or can secrete them. The gene for this has 2 cleavage sites:
1) The gene is initially one long transcript and the first stop codon is spliced out, producing a transmembrane domain. This holds the antigen at the cell membrane.
2) A short transcript splice acceptor is lost and there is no cleavage of the stop codon - causes secreted antigen.
ALTERNATING START SITES - how do isoforms differ? How does initiation of transcription by ribosomes cause different isoforms? LEAKY SCANNING
Isoforms differ by their n terminus sequence (at the start of the protein!).
The AUG site starts translation - if a sequence isn’t perfect the ribosome scans past the first AUG sequence, and stops at the 2nd/3rd etc. note - if a cell has high eIF-4f levels, ribosomes favour the 1st AUG.
PRODUCES 3 ISOFORMS OF ONE GENE - called ‘leaky scanning’.
Regulated nuclear transport - what is it? Rev (HIV related protein) - how does Rev cheat the system
RNT makes sure that no unspliced mRNA leaves the nucleus.
the HIV virus works by RNA reverse transcriptase, and the DNA produced is injected into the genome. HIV causes production of Rev - this cheats the nuclear system by allowing unspliced mRNA to exit the nucleus - high levels of Rev are a bad sign.
THE NEED FOR REGULATED TRANSLATION? role of eIF-2 and eIF2b.
eIF2 is needed to bind to GTP and initiate ribosomal scanning on the mRNA. eIF2B is needed for dissociation of GDP from eIF2.
This is needed because when a cell is at resting stage (G0)/infected/ or lacking nutrition, translation must decrease and this system allows this.
UTRs (un translated regions) - what are they, where are they found, what are they used for?
3’ UTRs
Un translated regions of mRNA. Found in all mRNAs. Occur at the coding regions of the open reading frame, the 3 and 5 prime end.
They are useful in protein localisation as signals at UTRs target it to a region of the cell.
3’ UTRs cause stem loops to form and the mRNA to be captured by proteins.
FUNCTIONS OF UTRs
Protein localisation, regulation of translation, IRON HOMEOSTASIS;
UTRs in control of iron levels; aconitase, ferritin
Ferritin is a protein that stores iron in the cell thus reducing Fe levels.
The transferrin receptor recruits Fe to the cell thus inreaseing Fe levels.
At low Fe conc, aconitase (a cytosolic protein) binds to the UTR of the gene coding for ferritin mRNA, thus blocking its transcription, preventing storage of Fe and increasing Fe levels.
At high Fe conc, aconitase binds Fe in the cytoplasm, undergoes a confirmational change, ferritin mRNA is then translated and causes;
- TRANSLATION OF FERRITIN - DEGRDATION OF TRANSFERRIN MRNA
IRES - what are they, how do they work?
Internal ribosomal entry sites. Mimic the 5’ cap and polyA tail - allow more than one gene to be transcribed therefore.
Found commonly in viral DNA transcripts. By positioning one coding sequence downstream of the 5’ cap/5’UTR in an mRNA, and a second gene downstream of an IRES sequence it is possible to allow the expression of two genes from a single mRNA.
RNA stability - variation in mRNA half life? Why? how is this changed
eIF-4E? Dan?
mRNA half life varies due to the length of its polyA tail - this polyA starts at 300 nucleotides in length and is chewed down to 30 by EXONUCLEASE.
Re-adenylation of mRNAs can extend their half life.
Commonly seen in promoting factors for translation eg- eIF-4E.
Dan is a de-adenylating nuclease and competes with eIF-4E to bind.
