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Hebrews 1:1-2 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; ©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 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! ©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 Control of Gene Expression Cytoplasm Packaging Degradation DNA Transcription Transportation Modification RNA RNA Processing mRNA G G AAAAAA Nucleus Export Degradation etc. AAAAAA Translation ©2000 Timothy G. Standish Logical Expression Control Points Increasing cost DNA packaging Transcription RNA processing mRNA export mRNA masking/unmasking and/or modification mRNA degradation Translation Protein modification Protein transport Protein degradation 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 Nucleosomes Metaphase Chromosome Tight helical fiber B DNA Helix Looped Domains Protein scaffold ©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 Initiation T. F. Promoter T. F. RNA Pol. II RNA Pol. II mRNA 5’ ©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 Transcription Start Site 5’ 5’ Untranslated Region Introns Exon 1 Int. 1 Promoter/ Control Region 3’ Untranslated Region Exon 2 3’ Int. 2 Exon 3 Exons Terminator Sequence RNA Transcript ©2000 Timothy G. Standish Eukaryotic Promoters Promoter 5’ Exon 1 Sequence elements TATA ~200 bp “TATA Box” Initiator Transcription start site SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN (Template strand) ~-25 -1+1 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 TFIID “TATA Box” Transcription start site TBP Associated Factors (TAFs) -1+1 TATA Binding Protein (TBP) ©2000 Timothy G. Standish Initiation TFIID Binding Transcription start site TFIID -1+1 80o Bend ©2000 Timothy G. Standish Initiation TFIIA and B Binding TFIID TFIIB Transcription start site -1+1 TFIIA ©2000 Timothy G. Standish Initiation TFIIF and RNA Polymerase Binding TFIID TFIIB Transcription start site -1+1 TFIIA TFIIF RNA Polymerase ©2000 Timothy G. Standish Initiation TFIIE Binding TFIIF TFIIB RNA Polymerase -1+1 TFIIA TFIIE TFIID Transcription start site 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 TFIIJ TFIIH TFIIF TFIIB P TFIIA PP RNA Polymerase -1+1 TFIIE TFIID Transcription start site TFIIH 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 TFIIJ TFIIH TFIIF TFIIB P PP -1+1 TFIIE TFIID Transcription start site RNA Polymerase TFIIA ©2000 Timothy G. Standish Initiation TFIIH and TFIIJ Binding Transcription start site P -1+1 PP RNA Polymerase ©2000 Timothy G. Standish Enhancers DNA Many bases 5’ 3’ Enhancer 5’ Promoter TF Transcribed Region 3’ TF 5’ TF TF RNA RNA Pol. Pol. 5’ 3’ RNA ©2000 Timothy G. Standish ©2000 Timothy G. Standish