©2000 Timothy G. Standish

Transkripsi

©2000 Timothy G. Standish

All Genes Can’t be Expressed At The Same Time

Some gene products are needed by all cells all the time. These constitutive genes are expressed by all cells.

Other genes are only needed by certain cells or at specific times, expression of these inducible genes is tightly controlled in most cells.

For example, pancreatic b cells make insulin by expressing the insulin gene. If neurons expressed insulin, problems would result.

©2000 Timothy G. Standish

3’

5’

5’

3’

Transcription And Translation In Prokaryotes

Ribosome

Ribosome5’

mRNA

RNAPol.

©2000 Timothy G. Standish

The mRNA Sequence Can Fold In Two Ways

4

1 23

Terminatorharipin

4

1 2

3

©2000 Timothy G. Standish

Expression Control In Eukaryotes Some of the general methods used to control

expression in prokaryotes are used in eukaryotes, but nothing resembling operons is known

Eukaryotic genes are controlled individually and each gene has specific control sequences preceding the transcription start site

In addition to controlling transcription, there are additional ways in which expression can be controlled in eukaryotes

©2000 Timothy G. Standish

Eukaryotes Have Large Complex Geneomes

The human genome is about 3 x 109 base pairs or ≈ 1 m of DNA

Because humans are diploid, each nucleus contains 6 x 109 base pairs or ≈ 2 m of DNA

Some gene families are located close to one another on the same chromosome

Genes with related functions appear to be distributed almost at random throughout the the genome

©2000 Timothy G. Standish

Highly Packaged DNA Cannot be Expressed

Because of its size, eukaryotic DNA must be packaged

Heterochromatin, the most highly packaged form of DNA, cannot be transcribed, therefore expression of genes is prevented

Chromosome puffs on some insect chomosomes illustrate areas of active gene expression

©2000 Timothy G. Standish

Only a Subset of Genes is Expressed at any Given Time

It takes lots of energy to express genes Thus it would be wasteful to express all

genes all the time By differential expression of genes, cells

can respond to changes in the environment Differential expression, allows cells to

specialize in multicelled organisms. Differential expression also allows

organisms to develop over time.

©2000 Timothy G. Standish

DNA

Cytoplasm

NucleusG AAAAAA

Export

Degradation etc.G AAAAAA

Control of Gene Expression

G AAAAAA

RNAProcessing

mRNA

RNA

Transcription

Nuclear pores

Ribosome

Translation

Packaging

Modification

Transportation

Degradation

©2000 Timothy G. Standish

Logical Expression Control Points DNA packaging Transcription RNA processing mRNA Export mRNA masking/unmasking

and/or modification mRNA degradation Translation Protein modification Protein transport Protein degradation

Increasing cost

Increasing cost

The logical place to control expression is before

the gene is

transcribed

The logical place to control expression is before

the gene is

transcribed

©2000 Timothy G. Standish

Three Eukaryotic RNA Polymerases

1RNA Polymerase I - Produces rRNA in the nucleolus, accounts for 50 - 70 % of transcription

2RNA Polymerase II - Produces mRNA in the nucleoplasm - 20 - 40 % of transcription

3RNA Polymerase III - Produces tRNA in the nucleoplasm - 10 % of transcription

©2000 Timothy G. Standish

A “Simple” Eukaryotic Gene

Terminator Sequence

Promoter/Control Region

Transcription Start Site5’ Untranslated Region

3’ Untranslated Region

Exons

Introns

3’5’ Exon 2 Exon 3Int. 2Exon 1Int. 1

RNA Transcript

©2000 Timothy G. Standish

5’DNA

3’

Enhancers

Enhancer Transcribed Region

3’5’ TF TFTF

3’5’ TF TFTF

5’RNA

RNAPol.

RNAPol.

Many bases

Promoter

©2000 Timothy G. Standish

Eukaryotic RNA Polymerase II RNA polymerase is a very fancy enzyme that does many tasks in conjunction with other proteins

RNA polymerase II is a protein complex of over 500 kD with more than 10 subunits:

©2000 Timothy G. Standish

Eukaryotic RNA Polymerase II Promoters

Several sequence elements spread over about 200 bp upstream from the transcription start site make up RNA Pol II promoters

Enhancers, in addition to promoters, influence the expression of genes

Eukaryotic expression control involves many more factors than control in prokaryotes

This allows much finer control of gene expression

©2000 Timothy G. Standish

RNA Pol. II

Initiation

T. F.

RNA Pol. II

5’mRNA

Promoter

T. F.

T. F.

©2000 Timothy G. Standish

Eukaryotic Promoters

5’ Exon 1Promoter

Sequence elements

~200 bp

TATA

~-25

InitiatorInitiator“TATA Box”“TATA Box”

Transcription start site

(Template strand) -1+1SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN

S = C or G Y = C or T N = A, T, G or C

©2000 Timothy G. Standish

InitiationTFIID Binding

-1+1

Transcription start site

TFIID

“TATA Box”

TBP Associated Factors (TAFs)

TATA Binding Protein (TBP)

©2000 Timothy G. Standish

InitiationTFIID Binding

TFIID

80o Bend

-1+1

Transcription start site

©2000 Timothy G. Standish

InitiationTFIIA and B Binding

TFIID

TFIIA

-1+1

Transcription start site

TFIIB

©2000 Timothy G. Standish

InitiationTFIIF and RNA Polymerase Binding

TFIID

TFIIA

-1+1

Transcription start site

TFIIB

RNA PolymeraseTFIIF

©2000 Timothy G. Standish

InitiationTFIIE Binding

TFIID

TFIIA

-1+1

Transcription start siteRNA Polymerase

TFIIBTFIIFTFIIE

TFIIE has some helicase activity and may by involved in unwinding DNA so that transcription can start

©2000 Timothy G. Standish

InitiationTFIIH and TFIIJ Binding

TFIID

TFIIA

-1+1

Transcription start siteRNA Polymerase

TFIIBTFIIFTFIIE

TFIIH has some helicase activity and may by involved in unwinding DNA so that transcription can start

TFIIH

P PP

TFIIJ

©2000 Timothy G. Standish

InitiationTFIIH and TFIIJ Binding

TFIID

TFIIA

-1+1

Transcription start siteRNA Polymerase

TFIIBTFIIFTFIIE

TFIIH

P PP

TFIIJ

©2000 Timothy G. Standish

InitiationTFIIH and TFIIJ Binding

-1+1

Transcription start site RNA Polymerase

P PP