Week 9 Flashcards
Transcriptional regulation
Determines how actively a gene is being transcribed, how many mRNA copies are being produced.
Control of transcriptional regulation
Promoter region
Chromatin status
Transcription factors
DNA methylation
Investigating gene expression transcription levels
Quantify mRNA: reverse transcription (qPCR), Northern blotting gene microarrays
Measure activity of promoter: Reporter gene assays (luciferase), chromatin immunoprecipitation.
qPCR (real time PCR)
Measures the amount of DNA that is present after each PCR cycle (using fluorescent DNA dye).
Northern blotting
Determines size of RNA transcript using gel electrophoresis after blotting.
Capping of mRNA
5’ end is methylated with guanosine
3’ end has a polyA tail and cleavage.
PAMPs
Pathogen associated molecular patterns, are expressed on innate immune cells.
Splicing
Removes introns from pre-mRNA and joins exons together. (Leaves mRNA with uninterrupted open reading frame). Occurs in the nucleus.
Splicing catalysts and regulators
snRNAa called spliceosomes regulate splicing, while U2, U4, U5 and U6 regulate splicing processes.
Tropomyosin
Regulates muscle cell contraction, alternatively spliced versions are expressed in different cells.
SR proteins
serve as positive regulators of splicing, binding to exon regions enhancing splicing eficiency.
hnRNP A and B
Bind to exon regions to suppress splicing.
Nova and Fox family proteins
Regulate splicing positively or negatively depending on where they bind (upstream or downstream) of the alternative exon.
Alternative splicing
Leads to the expression of different transcript variants, by leaving some genes in and cutting some out. This is normal, around 90% of genes are alternatively spliced.
Untranslated region (UTR)
Non-coding sequences on either side of the coding sequence of a messenger RNA molecule, specifically the 5’ and 3’ regions.
5 snRNPs and their functions
U1 recognizes the 5’ splice site
U2 binds to branch point (adenosine near 3’ end of intron)
U4, U5 & U6 join later to form the complete active spliceosome.
Steps of splcing
- U1 binds to 5’ site
- U2 binds to branch point
- U4, U5 & U6 joins the spliceosome
- U6 replaces U1, releasing U4
- A loop structure is made (branch point joins 5’ site)
- Intro in released
