Eukaryotic genes & the transcripts they produce (lecture 11)

Question 1

Bacterial gene organisation

Answer 1

Clustered genes are often transcribed as a single molecule of a mRNA

Such an mRNA is described as polycistronic

This allows coordinate expression of the gene cluster

Co-transcripted genes often encode proteins involved in the same process/biochemical

Question 2

What is polycistronic?

Answer 2

Multiple genes are transcribed as a single mRNA molecule

Question 3

What is monocistronic?

Answer 3

Each gene is a single transcription unit

Eukaryotic mRNAs are monocistronic

Since each gene has its own promotor, coordination of gene expression in eukaryotes is different to the situation in bacteria

Question 4

Key features of prokaryotic promotors

Answer 4

Conserved sequences upstream of the start site
• The conserved sequences are recognised by sigma factor
• This positions the polymerase allowing initiation of transcription
• RNA polymerase + sigma factor = holoenzyme

Bacteria only have 1 RNA polymerase

Question 5

What are the 3 different RNA polymerases in eukaryotes and what kind of genes do they transcribe?

Answer 5

1) RNA polymerase I – ribosomal RNA
2) RNA polymerase II – all protein coding genes
3) RNA polymerase III – tRNA, 5S rRNA & other small ‘non-coding RNAs’

Question 6

Key features of RNA polymerase II in eukaryotes

Answer 6

Promotor comprises various sequences (motifs) that are binding sites for factors that stimulate or repress transcription

Most important is the TATA-box
• TATA-box is bound by the transcription factor (TF) IID complex
• TFIID recruits other transcription factors & RNA polymerase 2

Question 7

Eukaryotic transcription termination

Answer 7

Transcripts end 10-35 nt downstream of a specific signal AAUAAA
Here, the RNA is cut by an endonuclease releasing it from the DNA
And the polyA tail is then added

Question 8

How do prokaryotes alter gene expression?

Answer 8

Alternative sigma factors recognise different -35/-10 sequences
• These control sets of co-ordinately regulated promoters e.g. heat-shock, stationary phase grow

Mutation to a single sigma factor affects expression of the set of genes that it regulates

Question 9

How do eukaryotes alter gene expression?

Answer 9

Many different transcription factors bind motifs in the promoter

These influence (both + and -) the ability of RNA pol II to initiate transcription (it NEEDS more than the basic TATA-box and TFIID).

This allows many stimuli to regulate each promoter and hence gene

Question 10

How can you analyse patterns of transcription?

Answer 10

Microarray

DNA corresponding to each gene in the organisms genome is spotted into a slide (the microarray)

Question 11

Pre-mRNA splicing

Answer 11

Intron & exon boundaires contain conserved sequences that
• Define their limits
• Recruit machinery which removes introns from the RNA, the ‘spliceosome’

Mutation in the intron doesn’t affect the coding sequence of the gene but could affect splicing

Mutation in the exon alters the coding sequence, & is likely to affect the sequence of the protein that it codes for

Question 12

What alterations are made to eukaryotic mRNA?

Answer 12

Capping
Polyadenylation - polyA tail
Splicing

Question 13

What is a eukaryotic gene?

Answer 13

A locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions

Not all genes encode proteins – many are transcribed to yield functional RNAs

Protein-coding genes don’t just encode 1 protein as alternative splicing can yield multiple products from each gene

Question 14

How is translation initiated?

Answer 14

Ribosomes comprise 2 subunits – large & small

The small subunit is responsible for reading the mRNA – ‘finding’ the start of open reading frames (ORF) in mRNA & interpreting each codon (pairing to tRNA)

The large subunit houses the protein synthetic peptidyl transferase centre

Question 15

How is translation initiated in bacteria?

Answer 15

The Shine-Dalgarno sequence is recognised directly by the small ribosomal subunit RNA by base pairing

Question 16

What is the Shine-Dalgarno sequence?

Answer 16

Responsible for aligning the ribosome with the start codon

It is a ribosomal binding site in bacterial and archaeal messenger RNA, generally located around 8 bases upstream of the start codon AUG

The large subunit binds to the small subunit & translation then initiates on the downstream AUG
This ‘defines’ the start of an ORF

Question 17

What does the Shine-Dalgarno sequence do?

Answer 17

Allows independent translation of each ORF in a polycistronic mRNA

Mutation of an SD sequence – reduced translation of the OFR it is associated with, but not other ORFs in the mRNA

Question 18

How is translation initiated in eukaryotes?

Answer 18

Depends of the 5’ cap on the mRNA

Question 19

Are prokaryotes coupled or uncoupled?

Answer 19

Coupled

Translation can affect transcription

Question 20

Are eukaryotes coupled or uncoupled?

Answer 20

Uncoupled

Translation cannot affect transcription

Question 21

Transcription summary

Answer 21

• Relies on sequences in the DNA (the promoter) to which transcription factors bind to promote initiation of RNA synthesis
• Bacterial transcripts are frequently polycistronic whereas eukaryotic mRNAs are monocistronic
• Introns must be removed from most eukaryotic pre-mRNAs to generate the functional mRNA (SPLICING)

Question 22

Translation summary

Answer 22

• Starts on a METHIONINE codon
• In prokaryotes this also relies on recognition of a specific signal, the Shine-Dalgarno sequence
• In eukaryotes the CAP on the mRNA is critical, and the first AUG in the mRNA is then used to initiate translation