regulation of gene expression in eukaryotes: post-transcriptional and post translational regulation

much of the processing of pre-mRNA occurs during (?)

transcription

capping

• addition of a methyl cap to the 5’ end of the mRNA

• occurs during transcription

polyadenylation

• addition of a poly-A tail to the 3’ end of the mRNA

• after mRNA synthesis has finished

splicing

• removal of introns

• usually during transcription but can occur post-transcription

capping factors attach a CAP structure to the 5’ end of the mRNA when about (?) nucleotides of the mRNA 5’ end are synthesised

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5’ cap contains a 5’-5’ (a) bridge between a methylated (?) and the 5’ end of the mRNA transcript

(a) triphosphate bridge

(b) guanosine

CAP functions

• signals correct 5’ end of the mRNA

• assists export of mRNA from the nucleus

• required for initiation of translation

• protects mRNA from 5’ degradation by RNases

splicing requires splicing sequences in the mRNA (list 2)

• intron/exon boundary sequences

• branch point A sequences in the intron

RNA complexes called (?) carry out the splicing

small nuclear ribonucleoproteins (snRNPs)

what is the type of RNA in snRNPs?

• small nuclear RNAs (snRNAs)

• snRNAs in snRNPs can base pair with the splicing sequences in the mRNA

two step mechanism of splicing (explain)

step 1

1. the branch point adenine in the intron has no base pair to the snRNA

2. it attacks the 5’ splice exon intron junction (5’ splice site) → cuts the sugar phosphate backbone of the RNA at this point

3. the released 5’ end of the intron becomes covalently linked to the 2’ -OH group of the ribose of the adenine nucleotide to form a branched structure

step 2

1. the free 3’-OH end of the released exon reacts with the start of the 3’ splice exon intron junction

2. this joins the two exons together into a continuous coding sequence

3. the intron is released as a lariat structure → eventually degraded in the nucleus

the three most important snRNPs for splicing are:

U1, U2 and U6

U1

recognises the 5’ splice site

U2

recognises the lariat branch-point site through complementary base pairing between snRNAs and mRNAs

U6

• double-checks the 5’ splice site by displacing U1 and base-pairing with the same intron sequence (5’ splice site) itself

• this improves the accuracy of splicing

conformational changes in (list 2) drive the formation of the spliceosome active site → splicing occurs

U2 and U6

after splicing the spliceosome deposits RNA-binding proteins (aka?) on the mRNA

• exon junction complex

• this marks the splice site as successfully completed

some pre mRNAs are (?) spliced by: (list two forms)

• alternatively

1. inclusion or exclusion of exons (or introns)

2. using alternative splice sites in exons

alternative splicing results in a number of different (?) from one gene

mRNAs → increases diversity of proteins

fruit flies regulates (?) splicing to generate stage-specific protein isoforms, a key mechanism for development, sex determination, neuronal wiring and muscle function

• alternative

• for example, alternative splicing of the Tra protein determines if a functional Tra protein is made → required to make a female fruit fly

the regulation of alternative splicing requires some regulatory proteins that bind to sequences in introns or exons → this binding interferes with binding of (?) thereby preventing splicing at a splice site

snRNPs

our genome has only about 20,000 protein coding (?)

• genes

• introns make up about 26% of the human genome

humans produce between 80,000 and 400,000 different proteins, despite only having 20,000 protein coding genes, due to (?)

alternative splicing

polyadenylation components (list 3: protein, factor, enzyme)

1. cleavage and polyadenylation specificity factor (CPSF)

• cleaves 3’ end of the pre-mRNA during transcription

2. poly-A-polymerase (PAP)

• recognises poly A signal on the mRNA

• binds about 10 nucleotides behind the signal and adds hundreds of adenines

3. poly-A-binding proteins

• assembles on poly-A-tail and determines final poly-A-tail length

• protects mRNA 3’ end

polyadenylation protects the mRNAs (a) end from degradation by (b)

(a) 3’ end

(b) RNase

proteins that bind to the mRNA after processing for export to the cytosol (list 3)

• CAP-binding protein

• poly-A-binding proteins

• exon junction complex

these proteins interact with export proteins that allow mRNA export through the (?) complex

nuclear pore

translational control in eukaryotes (explain)

• CAP binding protein is exchanged for elongation factor eIF4

• translation IFs, tRNA with methionine and the small ribosomal SU (SSU) assemble

• loaded SSU binds to mRNA 5’ CAP

• SSU scans the mRNA for an AUG

• the large ribosomal SU binds, translation initiation factors dissociate

• 3’ and 5’ mRNA loop forms

polyribosomes

several ribosomes bind to one mRNA to synthesise many copies of the same protein

mRNAs are degraded in the nucleus or cytosol if:

1. they are not correctly processed

• lack one of the three proteins that assist export (CAP binding protein, poly-A-tail binding protein, exon junction complex)

2. they are not needed anymore

eukaryotic mRNA half life span

several minutes to over 24 hours

degradation of the poly-A-tail is via what enzyme?

3’ to 5’ exonucleases

decapping enzyme removes the 5’ cap followed by degradation of mRNA in 5 to 3’ direction by what enzyme?

5’ to 3’ exonucleases

post-translational modification

covalent modifications of amino acids of a protein after its biosynthesis

PTMs diversify and specify protein function by: (list 4)

• promoting protein folding

• improving protein stability

• regulating protein activity

• targeting protein to the cell membrane

proteins to be degraded are marked by covalent addition of several copies of a protein called:

ubiquitin

the proteasome (multiprotein complex): what does it do?

• recognises polyubiquitin marked proteins

• marked proteins are unfolded by the stopper and channeled into the central cylinder of the proteasome

• inside the cylinder are several proteases that degrade the marked protein

name the 7 steps of regulation of gene expression in eukaryotes