Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Eukaryotes: similar 5 core subunits additional subunits E. coli RNA Polymerase Overall structure ~ DNA polymerase

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Eukaryotic Transcription Uses Multiple RNA Polymerases Eukaryotic promoters and consensus sequences are more diverse than those of bacteria Eukaryotes have three different RNA polymerases that recognize different promoters and produce different types of RNAs (you need to know each kind) The complex that assembles to initiate and elongate transcription is more complex in eukaryotes than in bacteria 2

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Eukaryotic Polymerases RNA polymerase I (RNA pol I) transcribes three ribosomal RNA genes RNA polymerase II (RNA pol II) transcribes protein coding genes and most small nuclear RNA genes (snRNA’s are involved in RNA “processing”) RNA polymerase III (RNA pol III) transcribes tRNA, one small nuclear RNA, and one ribosomal RNA 3

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Consensus Sequences for RNA Polymerase II Transcription To start transcribing DNA, the RNA polymerase must “recognize” a place on the DNA to which it should bind = promoter The promoters for RNA pol II genes are highly diverse with different overall lengths and number and types of consensus sequences 4

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Identifying promoters Find the DNA that encodes proteins = protein-coding genes (e.g. the genes that encode “histone” proteins = proteins used to wrap DNA and form chromatin) Compare DNA sequences in and around these genes – looking for shared sequence 5

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 6 Where is “upstream” of the gene?

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Promoter Elements The first-identified eukaryotic promoter consensus sequence was the TATA box, or the Goldberg- Hogness box, located at about position  25 The consensus sequence is 5-TATAAA-3 A CAAT box is often found near the -80 position A GC-rich box (consensus 5-GGGCGG-3) is located at  90, or further upstream 7

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 8 Which of these two strands is the CODING strand? How do you know? What does +1 signify?

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 9

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Promoter Elements How prove these “consensus” sequences have a function? Clone a gene and introduce it into an appropriate cell and ask if it can be expressed Modify the consensus sequences in the promoter and ask if the gene can STILL be expressed 10

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Promoter Elements 11

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 12 No promoter promoter single mutation in promoter x Plasmid (reporter gene is enzyme) Cell line in culture x x Transfect cell line with plasmid Add chromogenic substrate for enzyme nucleus

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 13 Myers et al. (Maniatis group) 1986: mutating beta-globin gene promoter

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Promoter Recognition RNA pol II recognizes and binds to promoter sequences with the aid of proteins called transcription factors (TFs) TFs bind to regulatory sequences and interact directly, or indirectly, with RNA polymerase; those interacting with pol II are called TFII factors The TATA box is the principle binding site during promoter recognition 14

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 15 TFIIA TFIID (TBP 2 + TAF) – binds TATAA box 6 TFII’s are also known as General Transcription Factors (GTFs) “Pre-initiation complex” – ready to go, but… Pol II begins transcription at +1 site Is this +1 site = ATG? Why called “General” TFs? TFIIA TFIIA, pre-initiation complex GTF’s

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 16 Enhancers can be “upstream” or “downstream” of gene; can be flipped in orientation yet will still work (although not palindromic); Silencers – similar interaction with promoter, but opposite effect

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Transcription Factor Synthesis Synthesis of transcription factors (binding enhancers/silencers) is tightly regulated and different cell types and cells at different developmental stages have distinct arrays of transcription factors (“specific” vs “general” TFs). The availability of activated transcription factors can also be controlled through signal transduction pathways – a means for things OUTSIDE the cell to tell the cell what to do (e.g. what genes to express) 17

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Receptor binds ligand (e.g. IL-12), associated Janus Kinases (JAKs) are activated to phosphorylate receptor and Signal Transducer and Transcription factors (STATs) that then dimerize and move to nucleus to bind promoters/enhancers of target genes An example of signal transduction – many other pathways possible

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach RNA Polymerase I Promoters RNA polymerase I transcribes genes for rRNA using a mechanism similar to that of RNA pol II Ribosomal genes are on chromosomal DNA, but they cluster together in a nuclear organelle called the nucleolus. The nucleolus is full of rRNA (product of Pol I transcription of rRNA genes). Ribosomes (protein and RNA) are assembled in nucleoli. 19

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 20 Ribosomal RNA genes: No TATAA box… but two Pol I-promoter “consensus” sequences Notice: Core element extends downstream of transcription start

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach RNA Polymerase III Promoters RNA polymerase III is primarily responsible for transcription of tRNA genes, but also transcribes one rRNA and other RNA-encoding genes (one snRNA, miRNAs, siRNAs) Small nuclear RNA genes (snRNA genes) have three upstream elements whereas the 5S rRNA and tRNA genes each have two internal promoter elements These are downstream of the start of transcription 21

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 22 5S rRNA and tRNA genes: “Internal Promoters” But same idea as “upstream promoters”: control elements bind proteins that position polymerase for transcription…

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Termination in RNA Polymerase I and III Transcription Transcription by RNA pol I (3 rRNA genes) is terminated at a 17-bp consensus sequence that binds transcription- terminating factor 1 (TTF1) – RNA is then cleaved 18bp upstream of this binding site RNA polymerase III transcription (tRNA) is terminated similarly to E. coli “intrinsic transcription termination” = long string of U’s (string of A’s in DNA) (A:U binding is not strong – polymerase falls off, DNA-RNA duplex falls apart) RNA pol II (protein-encoding genes) – termination is more complex… - post-transcriptional “processing” plays a role…

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Post-Transcriptional Processing RNA Pol II genes: The initial eukaryotic gene transcript is called the pre-mRNA whereas the fully processed form is called mature mRNA; modifications include 1. 5 capping 2. 3 polyadenylation 3. Intron splicing 24

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Capping 5 mRNA After the first 20 to 30 nucleotides of mRNA have been synthesized, a special enzyme, guanylyl transferase, adds a guanine to the 5 end of the pre-mRNA Additional enzyme action methylates the newly added guanine and may also methylate nearby nucleotides of the transcript The addition of the guanine to the mRNA is called 5 capping 25

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 26 N N NH 2 N O = 8 ribose CH 3 9 NH “m 7 G”

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Functions of the 5 Cap 1.Protects mRNA from rapid degradation (remember what a 5’ exonuclease recognizes?) 1.Facilitates subsequent intron splicing 1.Facilitates transport of mRNA out of the nucleus 1.Enhances translation efficiency by orienting the ribosome on the mRNA 27

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Polyadenylation of 3 Pre-mRNA The 3 end of the pre-mRNA is created by enzyme action that removes a section of the 3 message and replaces it with a string of adenines (no TTTT template in the DNA) A.AAUAA (polyA signal sequence in pre-mRNA) recognized by Cleavage & Polyadenylation Specificity Factor (CPSF) B.Cleavage Stimulating Factor (CStF) binds U-rich region downstream – recruits other factors, including Poly Adenylate Polymerase (PAP) C.Pre-mRNA is cleaved 15-30nt downstream of AAUAA (between AAUAA and U-rich region) & PAP adds a string of A’s (PABII’s facilitate and extend addition of A’s) 28

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 29 3’ UTR

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Torpedo RNAse degrades uncapped 3’ end of pre-mRNA Do not let diagram confuse you – 3’ end cleavage is 15-30nt downstream of AAUAA consensus

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Functions of Polyadenylation 1.Facilitating transport of mature mRNA across the nuclear membrane to the cytoplasm 2.Protecting the mRNA from degradation 3.Enhancing translation by enabling the ribosomal recognition of mRNA Note: Some eukaryotic transcripts (e.g., the histone genes) do not undergo polyadenylation 31

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Pre-mRNA Intron Splicing Intron splicing requires great precision to remove intron nucleotides accurately Errors in intron removal would lead to incorrect protein sequences (when exons are joined, the message needs to remain “in frame”) Roberts and Sharp shared the 1993 Nobel Prize for their codiscovery of “split genes,” i.e., the presence of intron and exon sequences 32

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach R-Looping The presence of intron sequences can be demonstrated by a technique called R-looping DNA encoding a gene is isolated and hybridized to mature mRNA from the same gene Regions of the DNA where introns are present have no complementary region within the mRNA, and thus loop out visibly 33

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach R-Looping 34 dsDNA mRNA 95 o C Template DNA anneals to mRNA

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 35

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Splicing Signal Sequences 36 Splicesome: “small nuclear ribonuclear proteins” = RNA + protein = SnRNPS U1, U2, U4, U5, U6 BRANCH SITE: nucleotides upstream of 3’ splice site Preferred mammalian: UACUAAC – does this match the consensus above? (N vs Y vs R) snRNP U1 binds 5’ splice site snRNP U2 binds branch site

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 37 U …CAGGUAAGU …CAG OH P GUAAGU… Exon1 intron Could DNA form a 2’-5’ phosphodiester bond? Exon 2Exon 1

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 38 ANIMATION: RNA Splicing

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Coupling of Pre-mRNA Processing Steps Introns appear to be removed one by one, but not necessarily in order The three steps of pre-mRNA processing are tightly coupled (what are these three steps?) The carboxyl terminal domain (CTD) of RNA polymerase II functions as an assembly platform and regulator of pre-mRNA processing machinery 39

© 2015 Pearson Education, Inc. 40

© 2015 Pearson Education, Inc.

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Mechanisms for Producing Alternative Transcript 1.pre-mRNA can be spliced in alternative patterns in different cell types (70% of human genes; less common in other animals and in plants) 2.Alternative promoters can initiate transcription at distinct start points 3.Alternative localizations of polyadenylation can produce different mature mRNAs Together these comprise alternative pre-mRNA processing WATCH “Alternative processing of pre-mRNA” movie on BLACKBOARD to understand this process 42

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach A note on nomenclature and gene drawings: pA site AAAAAAAAAAAAAAAAAAAAAA EXONS 5’UTR Pre-mRNA pA site AAAAAAAAAAAAAAAAAAAAAA EXONS 5’UTR GENE AUG UAA 3’UTR mRNA Yellow box is a non-coding exon… How many introns?