Transcription

Nicky Mulder

Acknowledgements: Anna Kramvis for lecture material (adapted here)

Copyright-Anna Kramvis 2

Central dogma of molecular biology

http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htmhttp://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm

DNA -> RNA -> PROTEIN

GENES are transcribed/expressed

Converts information from DNA into a usable template for protein

Copyright-Anna Kramvis 4

What is a gene?

Segment of DNA that is transcribed into RNA

This RNA can be:Non-coding –rRNA, tRNA, siRNA,

antisense RNAProtein coding –messenger RNA

(mRNA)

Copyright-Anna Kramvis 5

Parts of genes

The part of a coding gene that is translated into a protein is the Open Reading Frame (ORF)

Within the ORF, triplets of bases (codons) code for amino acids via the genetic code

An ORF starts with an initiation codon and ends with a stop codon

Prokaryotic genes

Promoter region

-35 -10

Regulatory regions

Open reading frame

Initiation site

Ribosome binding site

Termination site

Prokaryotic genes cont.

Can be encoded on different strands: forward or reverse

Promoter

Some are in operons

One transcript

Eukaryotic genes Many eukaryotic genes include introns and

exons Coding part is in the exons which need to

be joined

3’3’

exonexon exonexonintronintron

open reading frameopen reading frame

upstreamupstream downstreamdownstream5’5’

initiation codoninitiation codon termination codontermination codon

Eukaryotic gene reality

Small exons, large introns!

Exons are joined by splicing –can lead to multiple products

Alternative splicing

Exon 1 Intron 1 Exon 3Exon 2 Intron 2

Exon 1 Exon 2 Exon 3

Exon 3

Exon 3

Exon 1Exon 2

Exon 2

Splice junction

Transcription and translation steps

ProkaryotesEukaryotes

Why the RNA step?

MASTER COPY

FLEXIBLE & DISPENSABLE

Transcription

FINDING PROMOTER

INITIATION

ELONGATION

Transcription initiation

RNA Polymerase enzymes

Prokaryotes:

1 RNA polymerase, 4 subunits , , ’,

Eukaryotes:

3 different RNA polymerases, I, II, III -each 12-16 subunits

II is most well studied

Prokaryotic RNA Polymerase

SIGMA FACTOR IS IMPORTANT FOR

PROMOTER FINDING AND BINDING BY

RNA POLYMERASE

Eukaryotic RNA Polymerase II

Transcription initiation from RNA Polymerase II

Regulation of transcription -activation

PROMOTER

RNA POLYMERASE

/TF

ENHANCER

CO-ACTIVATOR

ADDITIONAL TRANSCRIPTION

FACTORS

ENHANCER PROTEIN

Regulation of transcription -repression

PROMOTERRNA

POLYMERASE

/TF

ENHANCER

ANTI- FACTOR

REPRESSOR

REPRESSOR

REPRESSOR

RBS

-HISTONE DEACETYLATION

-DNA METHYLATION

-CHROMATIN STRUCTURE

-AUTOREGULATION

Sigma factors

NH2 COOHCORE BINDING REGION

-10 -35

Transcription factors have DNA binding region and protein interaction domain

Sigma factor structure

RNA POLYMERASE

MAJOR FACTORS -HOUSEKEEPING GENES

RNA POLYMERASE

STRESS RESPONSE

EXTRACYTOPLASMIC

OTHER FUNCTIONS

ALTERNATIVE FACTORS

Regulation of factors

Autoregulation of its own expression Regulation of expression by external

signals Half-life of RNA or protein Relative abundance of different factors Regulation by anti-sigma factors Proximity of the sigma factors

Regulation by sigma factorsSPORULATION IN BACILLUS

HEAT SHOCK RESPONSE

Transcription and drug targets

RNA polymerase subunit is target for anti-TB drug rifampicin

SigB regulates katG- involved in INH resistance

Other transcription factors that have a core role in the control of expression of specific sets of genes eg IdeR

Gene regulation in humans

NF-KB

TP1

AP-1

STAT

Multicellular organisms need intra-cellular signals controlled by gene expression. Up to 10% of human genes may encode TFs.

Transcription regulation and human disease

Heart failure - Switch between 2 different metabolic pathways

Huntington’s disease - polyglutamine stretches & transcription dysregulation

Cancer - oncogenes and anti-oncogenes overexpression of genes, P53

Gene expression is NB

Having the right proteins at the right time No waste in producing what you don’t

need Being able to respond quickly to changes Getting the right regulators present to

control gene expression

Additional features of RNAs

Have ability to fold into secondary structures

RNAs can be catalytic –ribozymes RNAs can regulate transcription or

translation RNAs can be genomes

Antisense RNAs

Complementary to a piece of mRNA Bind and prevent translation Important form of post-transcriptional

regulation