Transcription: Chapter 13 Sections 13.1, 13.2, 13.4-13.13.8, 13.10-13.12 Transcription: Chapter 13

Transcription: Chapter 13 What is Transcription? The process of creating a transcript (RNA) from a template (DNA) In Eukaryotes, this process occurs in the nucleus and is complex. This process is separate from Translation. In Prokaryotes, this process occurs in the cytoplasm of the cell and is less complex. Transcription is often coupled with Translation. Transcription: Chapter 13

Transcription: Chapter 13

Key Players of Transcription RNA Polymerase Promoter Region Ribonucleoside Triphosphate Template Rho (r) Transcription Factors Cis -acting factors Trans acting factors Transcription: Chapter 13

How Do We Know That RNA Is The Intermediate Between DNA and Proteins? DNA is located in the nucleus. Proteins associated in the cytoplasm with ribosomes. RNA is chemically similar to DNA. RNA leave the nucleus and migrates to cytoplasm. RNA is proportional to proteins present in the cytoplasm Transcription: Chapter 13

Transcription (Prokaryotes) RNA Polymerase RNA polymerase (unwinds double helix) Ribonucleoside triphophates (U instead of T) No primer No proofreading abilities Ribose (not deoxyribose) Transcription: Chapter 13

Transcription: Chapter 13 Points to Ponder Can you draw deoxyribose and ribose? Can you differentiate Uracil from Thymine? Why is DNA considered a general and RNA a soldier? Can your draw a ribonucleoside triphosphate and a ribonucleoside monophosphate? Which one is the precursor for transcription? Transcription: Chapter 13

Transcription in Prokaryotes Transcription: Chapter 13

Transcription (Prokaryotes) Prokaryotic gene is divided into three parts Promoter RNA-coding sequences is the DNA sequence transcribed by RNA polymerase into the RNA transcript Terminator Transcription: Chapter 13

Transcription: Chapter 13

Transcription (Prokaryotes) RNA Polymerase (E. coli) – a, b, b’ s subunits (500,000 Da) b and b’ active site for transcription Sigma (s) subunit initiates RNA transcription by identifying promoter After 8 – 9 nucleotides assemble, s factor dissociates from complex Most common s subunit is s70 (s32, s54, ss, sE do exist) Transcription: Chapter 13

Promoter Region (Prokaryotes) TATAAT Box (-10 Region) – Pribnow box; cis acting factor TTGACA (-35 Region) – GAC box; cis acting Transcription: Chapter 13

Promoter consensus sequences TTGACA N16-17 TATAAT N6-7 A (+1) ... gene -35 box -10 box Start TTTACA N17 TATGTT N6 A (+1) ... gene Lac operon TTGACA N17 TTAACT N7 A (+1) ... gene Trp operon TTGATA N16 TATAAT N7 A (+1) ... gene RecA TTCCAA N17 TATACT N6 A (+1) ... gene LexA From: Fig. 12.4; Genetic Analysis and Principles 2nd Ed; Booker Transcription: Chapter 13

Transcription: Initiation in E. coli RNA Polymerase denatures DNA beginning at the -10 box 17 bp are denatured; forms transcription bubble Open promoter complex is formed (ssDNA) NTP’s begin to be inserted using +1 nt as the start site; sigma factor is released. Elongation begins Transcription: Chapter 13

Transcription: Chapter 13

Transcription: Elongation in E. coli RNA Polymerase denatures DNA as it moves RNA Polymerase continues to covalently bond NTP’s to the growing RNA strand RNA-DNA hybrid is formed for a short time after the RNA is produced; helps hold the complex together; structure in the RNA Polymerase separates the DNA-RNA hybrid; DNA re-anneals Rate of elongation is 30-50 nt/sec Transcription: Chapter 13

Transcription (Prokaryotes) Termination Rho (r)-independent termination – a sequence with two-fold symmetry/self-complementary about its center. Believed that the formation of the hairpin loop within the transcript causes destabilization of the RNA/DNA hybrid Transcription: Chapter 13

Transcription: Termination in E. coli Rho-independent termination or intrinsic termination Mediated by sequence elements Region that forms a stem loop followed by a uracil rich region near the end of the RNA After the RNA is synthesized, a stem loop forms Stem loop causes RNA pol to pause U-rich region binds to the DNA; unstable; causes dissociation of DNA-RNA hybrid Complex is release from the DNA; no protein required Transcription: Chapter 13

Transcription: Chapter 13

Transcription: Termination in E. coli Rho-dependent termination Rho (protein) binds to the rut (rho utilization site) on the newly synthesized RNA Rho moves toward the RNA pol A downstream portion of the newly synthesized RNA forms a stem loop Stem loop causes RNA pol to pause (contacts RNA pol) Rho continues to move along the RNA and catches up with RNA pol Rho acts as a helicase and breaks the H-bonds between the DNA-RNA hybrid; dissociation Transcription: Chapter 13

Transcription: Termination in E. coli Rho-dependent termination Fig. 10.4; Genomes 2 Transcription: Chapter 13

Transcription: Termination in E. coli Rho-dependant and Rho-independent termination Both involve a stem loop structure in the RNA Both involve the complete denaturation of the DNA-RNA hybrid and subsequent release of the RNA and RNA polymerase Transcription: Chapter 13

Transcription: Chapter 13 Points to Ponder Why does the sigma factor dissociates from the complex? What advantage does polycistronic RNA have in prokaryotes that monocistronic RNA does not have in eukaryotes? Why do you think ssBPs are not necessary in transciption but were vital in replication? Transcription: Chapter 13

Transcription in Eukaryotes Transcription: Chapter 13

Transcription (Eukarytotes) Three different types of RNA polymerases RNA polymerase I – located in nucleolus/rRNA RNA polymerase II – nucleoplasm/mRNA RNA polymerase III – nucleoplasm/tRNA/ 5S rRNA Transcription: Chapter 13

Transcription: Eukaryotes Other differences More complex (initiation and regulation) More proteins are need More regulatory elements in the DNA Extensive modification of the mRNA 3 different RNA polymerases Transcription: Chapter 13

Transcription: Chapter 13 Why do you think Transcription is more complex in Eukaryotes than Prokaryotes? Transcription: Chapter 13

Transcription: Chapter 13 Fig. 11.7 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes Transcription: Chapter 13 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Eukaryotic RNA polymerases RNA polymerases in Eukaryotes cannot bind directly to the DNA; all require protein transcription factors for initiation Larger than bacterial counterparts and have more subunits All 3 Eukaryotic RNA Polymerase have similar structure 2 large subunits—have homology to beta and beta prime found in prokaryotes (what activity do they have?) Transcription: Chapter 13

Eukaryotic RNA polymerases Synthesizes Location I rRNA (28S, 18S, 5.8S) nucleolus II All mRNAs and some snRNAs nucleoplasm III All tRNAs, 5S rRNA, other snRNAs Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation Promoters Basal promoter elements—components in the DNA that are needed to get transcription started at low levels (promote basal transcription) Proximal promoter elements—components that are a distance from the promoter, but play a role in enhancing or suppressing transcription; can influence the rate of transcription or whether a gene is transcribed. Transcription: Chapter 13

Transcription of mRNA in eukaryotes: Initiation Promoters Cis-acting (factors) elements—components of the promoter that are located in the DNA. (TATA box, CAAT box, GC box, enhancers, etc.) Trans-acting (factors) elements—components that bind to the DNA (protein transcription factors, RNA polymerase, etc.) Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation Promoters: Basal promoter elements TATA box (element) Found at approximately -25 Consensus sequence is 5’-TATAAAA-3’ Found in most eukaryotic promoters Thought to be important for locating +1; when mutated the start site is not set Important for basal transcription Analogous to the -10 box in prokyotes Initiator element (Inr)—pyrimidine rich region near +1 Transcription: Chapter 13

Can you think of 4 requirements for Basal-Level Transcription? Transcription: Chapter 13

ANSWER: RNA Polymerase, Transcription Factors, Proteins and Promoter Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation Promoters: Proximal promoter elements Usually upstream of the promoter (approx 50-200 bp) CAAT box Found at approx -75 GC box Found at approx -90 Can work in either orientation Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation Promoters: Regulatory elements—not required for basal transcription Cis-acting elements Can be near the promoter or located great distances from the promoter/gene May be upstream, downstream, or imbedded in the gene May be enhancers (activator proteins bind here) to increase the rate of or activate transcription; a single one or several May be silencers/suppressors (suppressor proteins bind here); decrease the rate of or stop transcription Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation RNA Polymerase cannot bind to the DNA, so transcription factors must bind first TFIID binds to the TATA box—multi-subunit TBP protein—binds TATA box TAF’s (at least 7)—binds TBP Binds to basal promoter elements; role similar to sigma factor in bacteria Transcription: Chapter 13

Transcription: Chapter 13 Fig. 11.5 Events that may occur during the initiation of transcription catalyzed by RNA polymerase II Transcription: Chapter 13 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Transcription of mRNA in Eukaryotes: Initiation TFIIB bind after TFIID is bound to the promoter B associated with D B recruits RNA pol II and TFIIF to the promoter TFIID, B, and F form the minimal promoter complex Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation TFIIE and H bind – makes initiation complex complete Produces the closed promoter complex TFIIH important in promoting the open promoter complex Using ATP, H phosphorylates the CTD (carboxyl terminal domain of RNA pol II Causes TFIIB to be released from the complex H then uses its helicase activity to denature the DNA TFIIE and H also dissociate when the open complex is formed Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation The described process of initiation leads to basal transcription To regulate gene expression and/or the rate of transcription, a protein called the mediator links transcription factors that bind to regulatory factors and RNA pol II Mediator thought to affect CTD phosphorylation Transcription: Chapter 13

Transcription of mRNA in Eukaryotes: Initiation Abortive transcription—a few nucleotides added in and transcription is stopped; not understood why this occurs Transcription: Chapter 13

Transcription: Chapter 13 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Transcription of mRNA in Eukaryotes: Elongation/Termination Transition to elongation when RNA pol becomes processive No termination signal/seq in euks; termination is part of the modification of the mRNA (addition of poly(A) tail at the 3’ end of the mRNA Transcription: Chapter 13

Transcription: Chapter 13

Genetic Code Characteristics RNA derived from DNA; Nucelotides used as “letters” 3 Nucleotides = 1 Codon = 1 amino acid 1 codon specifies 1 amino acid but 1 amino acid may have more than 1 codon Start and Start codons present No “breaks” between codons Transcription: Chapter 13

Genetic Code Characteristics At one location within the mRNA is a part of only one codon Code is universal Transcription: Chapter 13

Transcription: Chapter 13

Similarities between Prokaryotic and Eukaryotic mRNA Structure 5’ end—has a region that is not translated Called 5’ UTR (untranslated region) Variable length Regulatory 3’ end—has a region at the 3’ end that is not translated Called 3’ UTR (untranslated region) Transcription: Chapter 13

Similarities between Prokaryotic and Eukaryotic mRNA Structure Coding region—part of the mRNA that encodes the protein Contains an AUG in the proper context where translation starts Contains a stop codon in proper reading frame to end translation Length varies with the size of the polypeptide Transcription: Chapter 13

Similarities between Prokaryotic and Eukaryotic mRNA Structure Transcription: Chapter 13