©2001 Timothy G. Standish Psalm 102:24 25In the beginning you laid the foundations of the earth, and the heavens are the work of your hands.

©2001 Timothy G. Standish Initiation of Transcription: In Eukaryotes Timothy G. Standish, Ph. D.

©2001 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

©2001 Timothy G. Standish Eukaryotes Have Large Complex Geneomes The human genome is about 3 x 10 9 base pairs or ≈ 1 m of DNA Because humans are diploid, each nucleus contains 6 x 10 9 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

©2001 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

©2001 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.

©2001 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

©2001 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

©2001 Timothy G. Standish Three Eukaryotic RNA Polymerases 1 RNA Polymerase I - Produces rRNA in the nucleolus, accounts for % of transcription 2 RNA Polymerase II - Produces mRNA in the nucleoplasm % of transcription 3 RNA Polymerase III - Produces tRNA in the nucleoplasm - 10 % of transcription

©2001 Timothy G. Standish A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site 5’ Untranslated Region 3’ Untranslated Region Exons Introns 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 RNA Transcript

©2001 Timothy G. Standish 5’ DNA 3’ Enhancers EnhancerTranscribed Region 3’ 5’ TF 3’ 5’ TF 5’ RNA Pol. RNA Pol. Many bases Promoter

©2001 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:

©2001 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

©2001 Timothy G. Standish RNA Pol. IIInitiation T. F. RNA Pol. II 5’ mRNA Promoter T. F.

©2001 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

©2001 Timothy G. Standish Initiation TFIID Binding -1+1 Transcription start site TFIID “TATA Box” TBP Associated Factors (TAFs) TATA Binding Protein (TBP)

©2001 Timothy G. Standish Initiation TFIID Binding TFIID 80 o Bend -1+1 Transcription start site

©2001 Timothy G. Standish Initiation TFIIA and B Binding TFIID TFIIA -1+1 Transcription start site TFIIB

©2001 Timothy G. Standish Initiation TFIIF and RNA Polymerase Binding TFIID TFIIA -1+1 Transcription start site TFIIB RNA Polymerase TFIIF

©2001 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 by involved in unwinding DNA so that transcription can start

©2001 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 by involved in unwinding DNA so that transcription can start TFIIH P P P TFIIJ

©2001 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

©2001 Timothy G. Standish Initiation TFIIH and TFIIJ Binding -1+1 Transcription start site RNA Polymerase P P P

©2001 Timothy G. Standish