Upload
clemence-rich
View
212
Download
0
Embed Size (px)
Citation preview
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)