Eucaryotic Gene Expression

1. Eucaryotic Gene Expression Lecture 15

2. Key Eucaryotic Features • DNA is wrapped around chromatin • Nucleosomes made of histone proteins • Tightly packaged and organised into chromosomes • Relatively inaccessible • DNA lives in the nucleus • mRNA has to be transported out for translation • spatial and temporal separation of mRNA synthesis from protein synthesis • ribosomes attached to endoplasmic reticulum • DNA generally very big • Lots of genes • >30,000 in humans • Organelles have some of their own DNA • mitochondria, chloroplasts

3. Transcription • Three types of RNA polymerase • I for rRNA • II for mRNA • III for tRNA and rRNA • All 10 subunits (some shared) • inhibited by alpha-amanitin – from toadstools • No equivalent of sigma • A much more complex way of recognising promoters! • Need to open up the DNA • Transcribing areas more prone to DNase digestion

4. RNA pol Bit to be transcribed TBP Promoter Enhancers or Silencers Anywhere CCAAT-box -80 TATA-box -20-30 Eucaryotic Promoters TAF Basic, general transcription factors TAF TAF TAF NFY INR Note all the protein-protein interactions (as well as the DNA-protein interactions) The activity of all the proteins can be modified. Many transcription factors are tissue and/or time specific A string of TF binding sites in the promoter is called a PROMOTER MODULE

5. RNA pol TBP CCAAT-box -80 TATA-box -20-30 So far away… yet so close Enhancers or Silencers TAF TAF TAF TAF NFY INR Bit to be transcribed Promoter

6. mRNA Processing • The mRNA made in the nucleus is manipulated before export into the cytoplasm • POST-TRANSCRIPTIONAL processing • Capping • Addition of a methyl-guanosine residue to the 5’ end • To aid stability and help in ribosome binding • Happens during transcription • Tailing • Addition of 10s (or even 100s) of As to the 3’ end • Function not known • Done by polyadenylate kinase • after recognising –AAAUAA- near the end • Splicing • Cutting out sections of the mRNA (introns) • Sewing the remaining portions (exons) back together • The introns just get degraded 5’ PPP P G Me

7. mRNA splicing • Some genes are 95% intron! • A large part of the human genome is intron • Done by a SPLICEOSOME • A mixture of RNA and proteins • snRNP – small nuclear ribonuclear proteins (SNURPs) • Precision of splicing is cruical! • A change in reading frame would be a disaster • Spliceosome recognises specific sequences at the intron/exon boundary exon 1 intron 1 exon 2 intron 2 exon 3 exon 1 exon 2 exon 3

8. Alternate Splicing • Putting together the exons in different ways • Using different promoters • Using different exons as ‘cassette’ • 25% of human genes alternately spliced • Allows tremendous variation in some parts of the protein but constancy in others P1 exon 1 intron 1 P2 exon 2 intron 2 exon 3 using P1 and P2 will give different proteins – both with exon 3 exon 1 intron 1 exon 2 intron 2 exon 3 intron 3 exon 4 exon 1 could be stitched together with exon 2, 3 or 4

9. Other mRNA changes • mRNA Editing • Changing the sequence after transcription! • Best example is the truncation of apoprotein B • A protein involved in lipid transport around the body • In liver, transcript produces a large protein • In intestine, CAA half way along the mRNA changed to UAA • A stop codon! • So a shorter protein is produced. • mRNA degradation or storage • Eucaryotic mRNAs are more stable than procaryotic transcripts • can even be bound to inhibitory RNAs • to earmark for degradation or storage

10. Eucaryotic Translation • Basic mechanism the same • No polycistronic mRNAs • Ribosomes • 80 S • 60S - rRNAs 28S, 5.8S, 5S • 40S - rRNAs 18S • protein content varies according to species

11. Eucaryotic Translation • Initiation does not involve fmet • but methionine is initiating codon • mRNA scanned for first AUG • mRNA binds at 5’ end though cap • And perhaps a Kozak sequence –AACAUGAG- • Post-translational modification also common • Cutting up the protein (especially sequences used to ‘tag’ the protein for transport to certain organelles or membranes • Phosphorylation, glycolysation and other covalent modifications

12. Inhibitors • Some drugs affect eucaryotic translation • Cycloheximide • But ricin is the most interesting protein synthesis inhibitor to learn about • http://en.wikipedia.org/wiki/Ricin • cancer magic bullet?

13. Variation • Transcriptional • Huge temporal control • Massive complexity of transcription factors • Post-transcriptional • Several levels of splicing • Editing • Inactivation • Stability • Post-translational • All means that 30,000 genes gives a lot more variation than you’d expect For a given region of the genome, humans and chimpanzees share at least 98.5% of their DNA. How many genes make a face?

14. Text Book • p156-161 Differences between eucaryotic and procaryotic transcription • but don’t learn the intron splice site consensus sequence  • Several parts of Chapter 9 (Translation) have reference to eucaryotic/procaryotic differences • p180 – ribosomes • p181 – scanning for start site on mRNA • p187 – 5’ cap • Chapter 12 – Regulation of Eucaryotic Expression • p249 -255 • but just the gist of Fig 12-2 • Section on Alternative Splicing p260 – 265 • p261 is good, but the figure on p262 is too detailed – similarly it’s not necessary to know all the different splicing models listed on p263 and Figure 12-5 - just try to get the feeling of how easy it is to shuffle the exons • For RNA Editing • Just the last paragraph on p265 • For RNA stability • Just the first paragraph on this section (bottom of p268)