©2000 Timothy G. Standish Hebrews 1:1-2 1God, who at sundry times and in divers manners spake in time past unto the fathers by the prophets, 2Hath 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;

©2000 Timothy G. Standish Gene Regulation - Eukaryotes Timothy G. Standish, Ph. D.

©2000 Timothy G. Standish 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

©2000 Timothy G. Standish Eukaryotes Have Large Complex Genomes The human genome is about 3 x 10 9 base pairs or ≈ 1 m of DNA Because humans are diploid, each nucleus contains 6 3 x 10 9 base pairs or ≈ 2 m of DNA That is a lot to pack into a little nucleus!

©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 Nucleus G AAAAAA Export Degradation etc. G AAAAAA Control of Gene Expression G AAAAAA RNA Processing 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 The logical place to control expression is before the gene is transcribed

©2000 Timothy G. Standish 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.

©2000 Timothy G. Standish Packaging DNA G C A T Protein scaffold Metaphase Chromosome Looped Domains Nucleosomes B DNA Helix Tight helical fiber

©2000 Timothy G. Standish 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

©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. IIInitiation T. F. RNA Pol. II 5’ mRNA Promoter T. F.

©2000 Timothy G. Standish 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

©2000 Timothy G. Standish A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site 3’5’ RNA Transcript Introns Exon 2Exon 3 Int. 2 Exon 1 Int. 1 3’ Untranslated Region 5’ Untranslated Region Exons

©2000 Timothy G. Standish Eukaryotic Promoters 5’ Exon 1 Promoter Sequence elements ~200 bp TATA ~-25 Initiator “TATA Box” Transcription start site (Template strand) -1+1 SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN S = C or G Y = C or T N = A, T, G or C

©2000 Timothy G. Standish 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

©2000 Timothy G. Standish Initiation TFIID Binding -1+1 Transcription start site TFIID “TATA Box” TBP Associated Factors (TAFs) TATA Binding Protein (TBP)

©2000 Timothy G. Standish Initiation TFIID Binding TFIID 80 o Bend -1+1 Transcription start site

©2000 Timothy G. Standish Initiation TFIIA and B Binding TFIID TFIIA -1+1 Transcription start site TFIIB

©2000 Timothy G. Standish Initiation TFIIF and RNA Polymerase Binding TFIID TFIIA -1+1 Transcription start site TFIIB RNA Polymerase TFIIF

©2000 Timothy G. Standish Initiation TFIIE Binding TFIID TFIIA -1+1 Transcription start site RNA Polymerase TFIIB TFIIF TFIIE TFIIE has some helicase activity and may be involved in unwinding DNA so that transcription can start

©2000 Timothy G. Standish Initiation TFIIH and TFIIJ Binding TFIID TFIIA -1+1 Transcription start site RNA Polymerase TFIIB TFIIF TFIIE TFIIH has some helicase activity and may be involved in unwinding DNA so that transcription can start TFIIH P P P TFIIJ

©2000 Timothy G. Standish Initiation TFIIH and TFIIJ Binding TFIID TFIIA -1+1 Transcription start site RNA Polymerase TFIIB TFIIF TFIIE TFIIH P P P TFIIJ

©2000 Timothy G. Standish Initiation TFIIH and TFIIJ Binding -1+1 Transcription start site RNA Polymerase P P P

©2000 Timothy G. Standish 5’ DNA 3’ Enhancers EnhancerTranscribed Region 3’ 5’ TF 3’ 5’ TF 5’ RNA Pol. RNA Pol. Many bases Promoter

©2000 Timothy G. Standish