Hebrews 1 1 God, who at sundry times and in divers manners spake in time past unto the fathers by the prophets, 2 Hath in these last days spoken unto us by his Son, whom he hath appointed heir of all things, by whom also he made the worlds;

Gene Regulation - Eukaryotes Timothy G. Standish, Ph. D.

Control Of Expression 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

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 3 x 109 base pairs or ≈ 2 m of DNA That is a lot to pack into a little nucleus!

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.

Control of Gene Expression DNA Cytoplasm Nucleus Nuclear pores Packaging Degradation RNA Transcription Modification Ribosome Translation Transportation G AAAAAA RNA Processing mRNA Degradation etc. G AAAAAA G AAAAAA Export

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 The logical place to control expression is before the gene is transcribed

Eukaryotic DNA Must be Packaged Eukaryotic DNA exhibits many levels of packaging The fundamental unit is the nucleosome, DNA wound around histone proteins Nucleosomes arrange themselves together to form higher and higher levels of packaging.

Packaging DNA Metaphase Chromosome Protein scaffold Nucleosomes Looped Domains Nucleosomes B DNA Helix Tight helical fiber G C A T Metaphase Chromosome Protein scaffold

Highly Packaged DNA Cannot be Expressed The most highly packaged form of DNA is “heterochromatin” Heterochromatin cannot be transcribed, therefore expression of genes is prevented Chromosome puffs on some insect chomosomes illustrate where active gene expression is going on

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:

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

Initiation T. F. Promoter RNA Pol. II T. F. T. F. RNA Pol. II 5’ mRNA

Eukaryotic RNA Polymerase II Promoters Eukaryotic promoters are made up of a number of sequence elements spread over about 200 bp upstream from the transcription start site In addition to promoters, enhancers also influence the expression of genes Control of gene expression in eukaryotes involves many more factors than control in prokaryotes This allows much finer control of gene expression

A “Simple” Eukaryotic Gene Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region Introns 5’ 3’ Exon 1 Int. 1 Exon 2 Int. 2 Exon 3 Promoter/ Control Region Terminator Sequence Exons RNA Transcript

Exon 1 5’ Eukaryotic Promoters Promoter TATA Sequence elements SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN S = C or G Y = C or T N = A, T, G or C ~200 bp Transcription start site “TATA Box” Initiator (Template strand) -1+1 ~-25

Response Elements Response elements are short sequences found either within about 200 bp of the transcription start site, or as part of enhancers Different genes have different response elements Binding of transcription factors to response elements determines which genes will be expressed in any cell type under any set of conditions

Initiation TFIID Binding TBP Associated Factors (TAFs) Transcription start site “TATA Box” -1+1 TATA Binding Protein (TBP)

Initiation TFIID Binding Transcription start site TFIID 80o Bend -1+1

Initiation TFIIA and B Binding Transcription start site TFIID TFIIB -1+1 TFIIA

Initiation TFIIF and RNA Polymerase Binding Transcription start site TFIID TFIIB -1+1 TFIIA TFIIF RNA Polymerase

Initiation TFIIE Binding Transcription start site TFIIE TFIID TFIIF RNA Polymerase TFIIB -1+1 TFIIA TFIIE has some helicase activity and may by involved in unwinding DNA so that transcription can start

Initiation TFIIH and TFIIJ Binding Transcription start site TFIIE TFIID TFIIH TFIIF RNA Polymerase TFIIB P -1+1 TFIIA TFIIH has some helicase activity and may by involved in unwinding DNA so that transcription can start

Initiation TFIIH and TFIIJ Binding Transcription start site TFIIE TFIID TFIIH TFIIF RNA Polymerase TFIIB P -1+1 TFIIA

Initiation TFIIH and TFIIJ Binding Transcription start site RNA Polymerase P -1+1

Enhancers 5’ 3’ 3’ 5’ TF TF 3’ 5’ TF DNA Many bases Enhancer Promoter Transcribed Region TF 3’ 5’ TF TF 3’ 5’ TF RNA Pol. 5’ RNA Pol.

The End