© 2014 Pearson Education, Inc. Chapter 13 Mechanism of Transcription

© 2014 Pearson Education, Inc. Mechanism of Transcription (Introduction) Up to this point, we have been considering maintenance of the genome, that is, how the genetic material is organized, protected and replicated. We now turn to the question of how that genetic material is expressed, that is, how the series of bases in the DNA directs the production of RNAs and proteins that perform cellular functions and define cellular identity.

© 2014 Pearson Education, Inc. Figure Transcription of DNA into RNA Transcription is, chemically and enzymatically, very similar to DNA replication RNA polymerase does not need primer RNA product does not remain base-paired to the template DNA Lack of extensive proof reading compared to DNA replication

© 2014 Pearson Education, Inc. Table 13-1 β and β’ are homologous to RPB1 and RP2 α I and α II are homologous to RPB3 and RPB11 Pol I: transcribe large rRNA Pol III: transcribe tRNA, some small rRNA, small nuclear RNA and 5S rRNA Pol IV and Pol V only in plant: transcribe small interfering RNA

© 2014 Pearson Education, Inc. Figure 13-2 Comparison of crystal structure of prokaryotic and eukaryotic RNA polymerase (a) RNA Pol core enzyme of T. aquaticus (b) RNA pol II of S. cerevisiae

© 2014 Pearson Education, Inc. Figure 13-2a Mg2+ (a) RNA Pol core enzyme of T. aquaticus

© 2014 Pearson Education, Inc. Figure 13-2b RNA pol II of S. cerevisiae

© 2014 Pearson Education, Inc. Figure 13-3 Phase of transcription cycle: initiation, elongation and termination

© 2014 Pearson Education, Inc. initiation Phase of transcription cycle:

© 2014 Pearson Education, Inc. elongation termination

© 2014 Pearson Education, Inc. Figure 13-4 RNA polymerase holoenzyme (Core plus Sigma factor) 2 and 4 regions of sigma recognize -10 and -35 regions of the promoter, respectively

© 2014 Pearson Education, Inc. Figure 13-5 Features of various bacterial promoters Although the vast majority of sigma bind to -10 and -35 box, the sequences are not identical.

© 2014 Pearson Education, Inc. Figure 13-6 Regions of sigma Helix-turn-helix motif (DNA binding motif) within region 4 of sigma

© 2014 Pearson Education, Inc. Figure 13-7 Sigma and alpha subunits recruit RNA Pol core enzyme to the promoter Transition from closed complex to open complex by binding of sigma of RNA Pol holoenzyme and promoter is called isomerization which does not require energy from ATP hydrolysis.

© 2014 Pearson Education, Inc. Figure 13-8 Recognition and melting of the -10 box element by sigma region 2 Flip out bases

© 2014 Pearson Education, Inc. Figure 13-9 NT(non template) channel T(template) channel

© 2014 Pearson Education, Inc. Figure During initial transcription, RNA Pol remains stationary and pulls downstream DNA into it self (Scrunching)

© 2014 Pearson Education, Inc. Promoter escape involves breaking polymerase-promoter interactions and polymerase core-sigma interaction Elongating polymerase is a processive machine that synthesizes and proofreads RNA.

© 2014 Pearson Education, Inc. Figure Template and transcript within RNA Pol elongating complex Elongating polymerase is a processive machine that synthesizes and proofreads RNA.

© 2014 Pearson Education, Inc. Pyrophosphorolytic editing: enzyme uses its active site, in a simple back reaction, to catalyze the removal of an incorrectly inserted nucleotide, by reincorporation of PPi. After reincorporation, the mismatched rNTP is removed. Two types of proofreading of RNA Pol

© 2014 Pearson Education, Inc. Hydrolytic editing: Polymerase backtracks by one or more nucleotides and cleaves the RNA products, removing error containing sequence. Stimulated by Gre factor, which enhance editing and serve as elongation stimulation factor. This is comparable to those imposed on the RNA Pol II by TFIIS in eukaryote. Two types of proofreading of RNA Pol

© 2014 Pearson Education, Inc. RNA polymerase can become arrested and need removing. To deal with this situation, the cell has machinery that remove the arrested polymerase and at the same time recruits repair enzyme, endonuclease Uvr(A)BC. Both polymerase removal and repair enzyme recruitment are performed by a single protein called TRCF (Transcription Repair Coupling Factor: has ATPase activity).

© 2014 Pearson Education, Inc. Transcription is terminated by signals within the RNA sequence Sequence called terminator trigger the elongating polymerase to dissociate from DNA. There are two types of terminators Rho-dependent and Rho-independent terminators

© 2014 Pearson Education, Inc. Figure Rho transcription termination factor Rho pulls RNA out of polymerase, resulting in termination; or Rho induces a conformational changes in polymerase, causing enzyme to terminate. Rho has specificity to bind rut sites (site for Rho utilization).

© 2014 Pearson Education, Inc. Figure Rho-independent terminator (intrinsic terminator) A:U is the weakest of all base pairs. RNA readily dissociate from DNA

© 2014 Pearson Education, Inc. Figure 13-14

© 2014 Pearson Education, Inc. Figure TRANSCRIPTION in Eukaryotes General transcription factors (GTFs) and RNA Pol II core promoter (GTFs) (RNA Pol II core promoter) Downstream core element (DCE) Downstream promoter element(DPE)

© 2014 Pearson Education, Inc. Figure Promoter escape requires phosphorylation of the polymerase “tail”

© 2014 Pearson Education, Inc. Four Distinct Preinitiation Complexes TFIID with help from TFIIA binds to the TATA box forming the DA complex TFIIB binds next generating the DAB complex TFIIF helps RNA polymerase bind to a region from -34 to +17, now it is DABPolF complex Last the TFIIE then TFIIH bind to form the complete preinitiation complex = DABPolFEH In vitro the participation of TFIIA seems to be optional TFIID (bind to DNA with assistance of TFIIA) 1 2 DNA region from -34 to +17 Model of Formation of the DABPolF Complex -34 to +17 region of DNA

© 2014 Pearson Education, Inc. GTFs for RNA polymerase II TFIID TBP } TAFs IIB IIA IIE IIF IIH helicase protein kinase TBP Inr IIB IIA Pol IIa IIF IIE IIH CTD of large subunit of Pol II Recognize core promoter Targets Pol II to promoter Modulates helicase Helicase CTD protein kinase Many GTFs are possible targets for activators of transcription

© 2014 Pearson Education, Inc. Model for the participation of general transcription factors(GTFs) in initiation, promoter clearance, and elongation IIH: phosphorylate the CTD (serine 5), helicase activity During elongation, phosporylate serine 2 IIE: cross link to transcription bubble region TEFb: phosphorylate CTD further, elongate mRNA IIE and IIH are required for promoter clearance with energy by ATP Expansion of bubble with helicase of IIH releases stalled RNA Pol II

© 2014 Pearson Education, Inc. The Versatility of TBP Genetic studies have demonstrated TBP mutant cell extracts are deficient in: –Transcription of class II genes –Transcription of class I and III genes TBP is a universal transcription factor required by all three classes of genes Required in transcription of at least some genes of Archaea, single-celled organisms lacking nuclei

© 2014 Pearson Education, Inc Model for the Interaction Between TBP and Promoters Subunit of TFIID

© 2014 Pearson Education, Inc. Figure TBP-DNA complex TBP binds to and distorts DNA using a beta sheet inserted into the minor groove.

© 2014 Pearson Education, Inc. TBP bends DNA ~80 o and forces open the minor groove.

© 2014 Pearson Education, Inc. Table 13-2 (TBP associated factors)

© 2014 Pearson Education, Inc. Figure TFIIB-TBP- Promoter complex TBP TFIIB

© 2014 Pearson Education, Inc. Figure Assemble of the pre-initiation complex: In vivo, transcription initiation requires additional proteins, including the mediator complex

© 2014 Pearson Education, Inc. Figure 13-20

© 2014 Pearson Education, Inc. Figure A new set of factors Z(P-TEFb and others) stimulates Pol II elongation and RNA proofreading (TFIIS) Elongation factors: P-TEFb, SPT5(NusG in bacteria), TAT-SF1

© 2014 Pearson Education, Inc. Figure 13-21a

© 2014 Pearson Education, Inc. Figure 13-21b

© 2014 Pearson Education, Inc. Figure 13-22

© 2014 Pearson Education, Inc. Figure 13-23

© 2014 Pearson Education, Inc. Figure 13-24

© 2014 Pearson Education, Inc. Figure 13-25

© 2014 Pearson Education, Inc. Figure 13-26

© 2014 Pearson Education, Inc. Figure 13-26a

© 2014 Pearson Education, Inc. Figure 13-26b

© 2014 Pearson Education, Inc. Figure 13-27

© 2014 Pearson Education, Inc. Figure 13-27a

© 2014 Pearson Education, Inc. Figure 13-27b

© 2014 Pearson Education, Inc. Figure 13-28

© 2014 Pearson Education, Inc. Figure 13-28a

© 2014 Pearson Education, Inc. Figure 13-28b

© 2014 Pearson Education, Inc. Question 13-14