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. Eucaryotic Promoters INR TATA- box -20-30 CCAAT- box -80 Enhancers or Silencers Anywhere TBP TAF NFY RNA pol Bit to be transcribed Promoter Basic, general transcription factors 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. So far away… yet so close Bit to be transcribedINR TATA- box -20-30 CCAAT- box -80 Enhancers or Silencers TBP TAF NFY RNA pol 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 PPP 5’ G Me P

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 1intron 1exon 2intron 2exon 3 exon 1exon 2exon 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 exon 1intron 1exon 2intron 2exon 3 P1P2 exon 1intron 1exon 2intron 2exon 3 using P1 and P2 will give different proteins – both with exon 3 intron 3exon 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/Ricinhttp://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)