Eucaryotic Gene Expression Lecture 15. Key Eucaryotic Features DNA is wrapped around chromatin...

Eucaryotic Gene Expression

Lecture 15

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

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

Eucaryotic Promoters

INRTATA-box

-20-30

CCAAT-box

-80

Enhancers or Silencers

Anywhere

TBP

TAF

TAF

TAF

TAFNFY

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

So far away… yet so close

Bit to be transcribedINRTATA-box

-20-30

CCAAT-box

-80

Enhancers or Silencers

TBP

TAF

TAF

TAF

TAFNFY

RNA pol

Promoter

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

PPP5’

GMe

P

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

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 1 intron 1 exon 2 intron 2 exon 3P1 P2

exon 1 intron 1 exon 2 intron 2 exon 3

using P1 and P2 will give different proteins – both with exon 3

intron 3 exon 4

exon 1 could be stitched together with exon 2, 3 or 4

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

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

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

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?

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?

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)