L11: Posttranacriptiona Regulation Of Eukaryotic Gene Expression

Question 1

Post transcriptional control of eukaryotic gene expression

Answer 1

Regulation of transcription initiation: principal mechanism controlling gene expression

Mechanisms operate after RNA pol has bound promoter and RNA synthesis has begun. Some are co-transcriptional

Multiple control points

Question 2

mRNAs never occur as free RNA molecules

Answer 2

Always have associated proteins

Ribonucleoprotein (RNP) complexes. Contain other RNAs. Involved in transcript processing steps. Components of RNP complexes change as mRNA is processed, leaves nucleus and when in cytoplasm

Question 3

Mature mRNA/ transcript= mature mRNP

Answer 3

mRNA with 5’ cap, poly(A) tail & introns removed

Ready to leave nucleus

If still in nucleus= nuclear mRNPs

If in cytoplasm= cytoplasmic mRNPs

If not processed correctly = degraded by nuclear exosomes (need cap, tail and associated proteins to be deemed ok)

Question 4

RNA pol II carboxy-terminal domain (CTD)

Answer 4

mRNA capping

10x longer than rest of pol II when extended

Docking site for numerous capping proteins

Couples RNA processing with transcriptional elongation

5’ cap: capping proteins (factors) bind when Ser5 residues in repeats are phosphorylated (late in transcription initiation)

Question 5

5’ cap

Answer 5

After ~25 bases of mRNA synthesised

Phosphatase removes 5’ phosphate

Guanyl transferase adds GMP in reverse linkage (5’ to 5’ linkage)

Methyl transferase adds methyl group to guanosine. Methyl group added to second ribose of some transcripts

Question 6

Cap-binding complex added

Answer 6

Protects 5’ cap from degradation

Question 7

Consensus sequence

Answer 7

Sequence representing most frequent residues at positions in sequence (determined from aligning sequences of same gene, gene region or genomic region or protein or protein region)

Question 8

Important consensus sequences in mRNA 3’ processing

Answer 8

All present in gene sequence encoding mRNA

AAUAAA: poly(A) signal. 10-35 nucleotides upstream of cleavage and poly(A) tail attachment site.

GU rich region beyond cleavage site

CA nucleotide pair at least 30 nucleotides upstream of GU rich region

Question 9

Poly-A pol (PAP)

Answer 9

Adds ~200 As to cleaved 3’ end -> 3’ end protected from exonucleases

Question 10

Intron splicing

Answer 10

For short transcripts with few introns: Occurs after 3’ end processing steps

For longer transcripts with multiple introns: occurs before transcription ends

Consensus sequences at 5’ and 3’ spice sites within intron. 5’ = GU & 3’ = AG

Splicing proteins associated with CTD of RNA pol II

Question 11

Intron splicing

Answer 11

Adenosine at branch point attacks 5’ splice site -> sugar-phosphate backbone cleaved. Cleaved 5’ end of intron covalently linked to adenosine -> lariat structure formed

3’ end of upstream exon reacts with 5’ end of next exon. Sugar phosphate backbones joined. Lariat intron sequence released -> degraded by nuclear RNases

Question 12

Alternative splicing

Answer 12

75% of human genes produce transcripts that are alternatively spliced

Increases coding potential of eukaryotic genomes. One gene doesn’t necessarily code for only one protein.

Some genes encode transcripts that show constitutive splicing. Same mature transcript made continuously by cells

Some genes encode transcripts that show cell specific and/or time specific alternative splicing

Splicing done by spliceosome. 5’= GU and 3’= AG recognised for each intron

Question 13

Regulation of Alternative Splicing

Answer 13

Dependent on gene regulatory proteins present

Negative regulation: repressor proteins block access of splicing machinery to particular splice sites on pre-mRNA.

Positive regulation: activator proteins bind specific nucleotide sequences. May be many nucleotides from splice site = splicing enhancers. Activator proteins directs splicing machinery to an otherwise overlooked splice site

Question 14

mRNA transport from nucleus

Answer 14

Only fully processed RNAs are exported. Incorrectly processed or damaged transcripts are degraded in nucleus (exosomes)

Particular hnRNP proteins must be associated with mRNA before it can be exported. Some dissociate before export through NPC some remain attached to exported mRNA. Entire set of hnRNP protein marks mRNA as being export ready or not. Nuclear export receptor (aka nuclear transport receptor) must be present -> complex of proteins and transcript can exit nucleus

Export through nuclear pore complex (NPC): ~30 different proteins (nucleoporins). H2O filled channel. Protein filaments extend into nucleoplasm & cytoplasm. In nucleoplasm -> form nuclear basket.

In cytoplasm: nuclear export receptor dissociates; reimported to nucleoplasm. Proteins are exchanged. E.g CBC exchanged for translation IF

Question 15

Mature mRNAs differ in their stability

Answer 15

Stable mature mRNAs persist after transcription of genes is repressed

Half life of prokaryotic mRNA: few mins (good thing. Need to react dynamically to where they are -> change expression patterns)

Half life of most mRNAs from multicellular eukaryotes = many hours (cellular environment more controlled, reliable & constant -> longer lived)

3’ UTRs carry info that control mRNA lifetimes. Binding sites for proteins involved in deadenylation, decapping and degradation -> exonucleolytic decay