Gene Regulation 6 - Regulation of eukaryotic gene expression - post transcriptional and post translational regulation Flashcards
Learning Outcomes
Students will be able to
▪ describe mRNA processing events and their outcomes and how these impact on mRNA
export and translation initiation.
▪ describe an mRNA degradation pathway.
▪ state what miRNAs are and how they can impact of gene expression.
▪ understand that PTMs can impact on protein function.
▪ describe the role of a proteasome in protein degradation.
Comparing a Gene and Its Transcript
Pre-mRNA processing - overview
- Primary transcript = pre-mRNA
undergoes extensive processing to
produce the mature, functional RNA
– 5’cap = 7-methyl guanosine linked
by 3 Phosphates
– Splicing - removal of introns
– Poly A tail (±200), polyA
polymerase
Co-transcriptional processing: 1) Capping
During transcription:
* ~ 25 nt of the mRNA 5’-end are synthesised.
* Capping factors (proteins) attaches a CAP
structure to the 5’-end of the mRNA.
* CAP: unusual structure containing 5’ to 5’
triphosphate bridge between the CAP and the 5’-
end of the mRNA.
* CAP functions:
– signals correct 5’-end of the mRNA ➔ required
for:
* Export of mRNA from the nucleus
* Initiation of translation
– Protection from 5’ degradation by RNases
Human genes can vary greatly in size
and numbers of exons and introns
- Average mammalian intron ± 2000 nt, average exon ±200 nt
– Human β-globin gene encodes for one of the protein subunits of the oxygen carrying
protein haemoglobin - 3 exons, 2000 bp = 2 kbp
– Human Factor VIII gene encodes blood clotting protein – 26 exons , 200000 bp = 200
kbp
– Human Dystrophin gene encodes for a skeletal muscle protein, largest known human
gene, 2200000 bp = 2200 kbp (0.1 of the human genome), 79 exons, takes 16 hours to
be transcribed, mutations are associated with Duchenne muscular dystrophy
Co-transcriptional processing: 2) Splicing
- Splicing removes introns as ‘lariat’ structures
– VERY precise - Requires splicing sequences
– intron/exon boundary sequences
– sequences in the intron - Splicing is carried out by interaction of
spliceosomes ‘snurps’ with splicing sequences
on the pre-mRNA.
– Spliceosomes are complexes of proteins &
small nuclear RNAs (snRNA).
– snRNAs hybridise with splicing sequences
Why do genes have introns ?
Intron maintenance in the genome = extra cost and energy
Is there a selective advantage justifying this?
▪ Drosophila melanogaster has fewer genes than Caenorhabditis elegans BUT: the protein
number does not reflect the lower gene number
WHY? - Alternative splicing
▪ Results in a number of different mRNAs from one gene ➔ increases diversity of proteins and
functionality
~ 95% of human genes with more than one exon are alternatively spliced
~ 20,000 protein coding genes (~2% of human genome) ➔ >80,000 proteins
▪ THUS one gene one protein is not always true, one gene can result in many proteins
▪ Many splicing events are rare, tissue specific, developmentally regulated
Co-transcriptional processing: 3) Polyadenylation
Cleavage and polyadenylation specificity factor (CPSF)
▪ cleaves 3’-end of the pre-mRNA from the mRNA still
being synthesized
Poly-A-polymerase (PAP)
* Recognises poly A signal on the mRNA (AAUAAA or
AUUAAA)
* binds about 10 nucleotides behind the signal and
adds many hundreds adenines (one at a time)
Poly-A-binding proteins (Pab)
* assemble on poly-A tail and determines final poly-A
tail length (unknown mechanism), protects mRNA 3’-
endCPSF
Polyadenylation protects the mRNA’s 3’ end from
degradation by RNase
mRNA export to the cytosol
➢ Proteins bound to mRNA after processing are required
for export to the cytosol
* CAP binding protein
* exon – exon junctions proteins
* Poly A binding proteins
➢ These proteins interact with export proteins that allow
mRNA export through the nuclear pore complex to the
cytosol.
➢ In the cytosol, some of the proteins are exchanged for
cytosolic variants before the mRNAs associates with
ribosomes.
* Example Cap binding protein is exchanged for eIF4
Translational Control is often exerted at the
initiation of translation stage
- 5’ cap binds to eIFs = eukaryotic
initiation factors. - Eukaryotes KOZAK consensus
sequence ACCAUGG (not shown)
around start codon is important
in binding small ribosomal
subunit.
– In prokaryotes: Shine-Dalgarno
sequence - Ribosome SSU assembles & slides
along mRNA finding translation
start codon AUG. - Loop between 3’ and 5’ mRNA
ends forms = Checkpoint for
intact mRNA ends. - 5’ and 3’-UTRs sequences specify
efficiency of translation.
mRNA half-life and degradation
- Eukaryotic mRNA half life: several minutes to over 24 hours
- mRNA sequences determine half life and mRNA degradation pathway
– Example: 3’-UTRs carries information to specify mRNA half-life - Degradation begins with poly-A tail shortening by a 3’ to 5’ exonuclease
– The 3’ to 5’ degradation can continue to degrade the whole mRNA - Decapping enzyme removes the 5’-cap
- Degradation of mRNA in 5’ to 3’ direction by 5’ to 3’ exonuclease
miRNA mediated RNA silencing and post-transcriptional
regulation of gene expression
- miRNAs ‘micro RNAs’: small noncoding RNA molecules (~ 22 nt)
- Encoded by nuclear DNA in plants
and animals and by viral DNA in
certain viruses - Incorporated into a protein
complex (RISC) - Function via base-pairing with
mRNA molecules:
➢ extensive sequence match
to mRNA: mRNA cleavage
➢ less extensive sequence
match with mRNA:
translational repression
Post-translational modification (PTM) of proteins
- Covalent modification of amino acids
or N- or C-terminus of a protein after
its biosynthesis - Modifications are done by enzymes,
e.g. phosphorylation by kinases -
most common PTM. - PTMs diversify and specify protein
function by:
– promoting protein folding
– improving protein stability
– regulating protein activity,
– targeting protein to the cell
membrane
Proteasome degrades short-lived and misfolded proteins
- Proteins to be degraded are marked by covalent addition of several proteins named ubiquitin
➔ polyubiquitin - Proteasome is a multiprotein complex:
– The proteasome stopper region recognises polyubiquitin marked proteins
– The marked proteins is unfolded by the stopper and channelled into the central cylinder of
the proteasome.
– Inside the cylinder are several proteases that degrade the marked protein
The Phenotype of a cell, tissue or organism
is determined by the Genotype BUT…..
- Genotype determines the phenotype ➔ the phenotype is the end product of gene expression
- BUT – there it is a long way from a genotype to a phenotype with many opportunities to
influence gene expression
Can you …
▪ … name and explain the workings and outcomes of mRNA processing mechanisms in eukaryotes?
▪ … can you explain alternative splicing and how this demonstrates that one gene one protein is often not
true?
▪ … can you name the main proteins involved in the export of a completely processed mRNA from the
nucleus to the cytosol?
▪ … can you describe how mRNA sequence motifs and proteins control the initiation of translation?
▪ … describe how mRNA degradation works and how it contributes to regulation of gene expression?
▪ … explain the basic workings of miRNAs in the regulation of gene expression?
▪ … give an overview of post-translational protein modifications and give examples oi their outcomes?
▪ … give an overview of the ubiquitin mediated degradation of proteins as an example of the regulation of
gene expression
