Nucleotide Entry Port. Scaffold Subunits. Polymerase Activity β Sliding Clamp. Clamp Loader. Promoter Recognition

Transcription

1 Nucleotide Entry ort α (2) Scaffold Subunits olymerase Activity Sliding Clamp σ Clamp Loader romoter Recognition

2 NNAAA AA T A TTTTNNAAAANNN TT T N N17 N6 α α α α α α α α α α α α α α +1

3 α α α α Subunit Function E coli Homologue RB1 polymerase activity CTD - ro-thr-ser- subunit ro-ser-tyr-ser RB2 clamp subunit RB3 scaffold, assembly α subunits RB4 helps to recognizes promoter σ subunit common to all RB5, RB6, RB8, eukaryotic RNA RB10, & RB12 olymerases RB3, RB4, & RB7 unique to ol II RB9, & RB11????

4 GGGCGG GGCCAATC TATAAAA Inr High Mobility Group roteins roximal Initiator Elements (Enhancers) Core romoter Elements or Basal romoter Elements (DNA Sequences) +1 GGGCGG GGCCAATC BRE TATA Inr DE % of 15% of 12.5% of % of Response Element Distal romoter Elements or Enhancers & Silencers TATAAA Inr TB TAF s TFIIA TB TATAAA TFIIA TFIIB TB TATAAA Inr TFIIB TFIIA

5 TFIIH TFIIF RNA ol II TB TATAAA Inr TFIIB TFIIA TFIIF RNA ol II TFIIE TB TATAAA Inr TFIIB TFIIA TFIIF RNA ol II TFIIE TFIIH Closed Complex TFIIF RNA ol II Inr TB TATAAA TFIIB TFIIA TFIIE TFIIH Open Complex hosphorylation of CTD of ol II by TFIIH and/or Other rotein Kinase Initiation Complete romoter Escape TFIIF RNA ol II TFIIA TB TATAAA TFIIB TFIIE TFIIH

6 RNA rocessing RNA rocessing trna rocessing Bacterial trna genes contain 2 to 5 trna s. Eukaryote trna each gene codes for 1 trna. In Bacteria RNase (endonuclease) cuts at mature 5 end. Mature 3 end produced by a combination of RNase & RNaseD (exonuclease) in Bacteria and Eukaryotes. All trna s have a 3 end of CCA. trna Nucleotidyltransferase adds CCA to 3 end of those trna s that do not have the sequence as part of the gene. trna rocessing (continued) Numerous Bases Modified Including 1-Methylguanosine Ribothymidine Inosine 4-Thiouridine Dihydrouridine seudouridine

7 rrna rocessing Bacteria contain 3 pieces of rrna. 5S (120 nucleotides), 16S (1540 nucleotides), & 23S (3200 nucleotides) contained on single gene. Eukaryotes contain 4 pieces of rrna. 5S (120 nucleotides), 5.8S (160 nucleotides) 18S (1900 nucleotides), & 28S (4700 nucleotides). 5S piece a single gene. The other three pieces on one gene. rrna rocessing (continued) Complex process Combination of endonucleases and exonucleases separate the pieces Specific bases methylated using SAM as methyl donor rrna pieces fold into complex three dimensional structures as they interact with / bind to ribosomal proteins

8 GU O O O O O O O CH Base 2 O O O O O O O O Ribonucleic Acid O 3 (1) hosphohydrolase 4 O O O O O CH Base 2 O O O O GT O O O Ribonucleic Acid CA Synthesizing Complex CA Binding Complex O 3 4 O 3 4 (2) Guanylyltransferase O HN N H 2N N N O O O H O CH O 2C O O O Base 2 O O O O O SAM O HN O O O Ribonucleic Acid (3) Guanine-7-methyltransferase adohcy CH 3 N H 2N N N O O O H O CH O 2C O O O Base 2 O O O O O O O O Ribonucleic Acid y-n-y-u-a-y GU Branch oint-binding rotein U1 U1 U1 U1 GU A U2 U1 U1 GU A

9 U1 U1 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAUAAA HO A GU A UG-O U6 U6 U2 U2 GU A U5 U5 U1 U1 U6 U6 U4 U4 U2 U2 GU A U5 U5 GU A U6 U4 U5 U1 U4 Cleavage & olyadenylation Specificity Factor (CSF) olyadenylate olymerase (oly A olymerase) Endonuclease Specificity & Activity Factors AAUAAA AAUAAA A UG-O

10 Why are there Introns? How did Introns arise? TATA1 DNA Exon A GT Intron 1 TATA2 Exon B GT Intron 2 Exon C Transcription hnrna if TATA1 Employed Exon A GU Intron 1 TATA2 Exon B GU Intron 2 Exon C Transcription hnrna if TATA2 Employed Splicing Exon A Exon C Exon B GU Intron 2 Exon C Splicing Exon B Exon C Exon Y GT DNA AATAAA Exon Z AATAAA Transcription Exon Y GU AAUAAA Exon Z AAUAAA If 1st olyadenylation Site Employed Exon Y GU AAUAAA AAAAAAAAAAAAAAAAAAAAAA If 2nd olyadenylation Site Employed Exon Y Exon Z AAUAAA AAAAAAAAAAAAAAA

11 cam R R R R CR cam CR

12 R R R R CR R R R R Control of Transcription in Eukaryotes Chromatin Remodeling Heterochromatin vs. Euchromatin Methylated G-C Sequences Z-form DNA Methylation of H3 Acetylation of Core Histones H3 & H4 Histone Acetyltransferases & Histone Deacetylases

13 Some terminology: ROMOTER ELEMENTS are regions of non-coding DNA that regulate the transcription of nearby genes; older nomenclature CIS-REGULATORY ELEMENTS. TRANSCRITION FACTORS are proteins that bind to the promoter elements to bring about the initiation of transcription or the genes that code for these proteins; older nomenclature TRANS- REGULATORY ELEMENTS. High Mobility Group roteins roximal Initiator Elements (Enhancers) Core romoter Elements or Basal romoter Elements (DNA Sequences) GGGCGG GGCCAATC BRE TFIIB TFIIF TATA TFIIATFIIB TB ol II +1 Inr TFIIE DE TFIIH Mediator Basal Transcription Factors {roteins that Bind to Core romoter Elements} DNA Binding Transactivators Signal Response Distal Element Upstream Initiator Elements Enhancers & Silencers Core and roximal Activation Sequences Sequence Module Consensus Sequence Locatio DNA Bound rotein Factor n TATA Box TATAAAA -25 ~10 bp TB BRE G/C G /C G /ACGCC -35?? TFIIB DE A/GG A /T C /T G / A /C +30???? CAAT Box GGCCAATCT -70 ~22 bp CTF/NF1 GC Box GGGCGG -110 ~20 bp S1

14 Distal Upstream or Downstream Activation Sequences Sequence Module Consensus Sequence DNA Bound rotein Factor GC Box GGGCGGG ~20 bp S1 Octamer ATTTGCAT ~20 bp Oct-1 Octamer ATTTGCAT ~23 bp Oct-2 κb GGGACTTTCC ~10 bp NFκB κb GGGACTTTCC ~10 bp H2-TF1 ATF GTGACGT ~10 bp ATF Response Elements to articular hysiological Challenges hysiological Challenge Response Element Consensus Sequence DNA Bound rotein Factor Size (kd) Heat shock HSE CNNGAANNTCCNNG 27 bp HSTF 93 Glucocorticoid GRE TGGTACAAATGTTCT 20 bp Receptor 94 Cadmium MRE CGNCCCGGNCNC??? horbol Esters TRE TGACTCA 22 bp A1 39 Serum SRE CCATATTG 20 bp SrF 52 rotein Kinase A Activation CRE TGACGTCA? CREB 43

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