Transcription

Ex Biochem c11-transcription Introduction Coding strand: identical in sequence with RNA Template strand: used as template for RNA synthesis Complimentary to RNA Figure 11.1

Ex Biochem c11-transcription 11.1 Introduction RNA polymerase Promoter: a special region containing startpoint Terminator Upstream: sequences prior to startpoint Downstream: sequences after startpoint Figure 11.2

Ex Biochem c11-transcription 11.2 Transcription Occurs by Base Pairing in a “Bubble” of Unpaired DNA RNA polymerase separates the two strands of DNA in a transient “bubble.” When RNA polymerase bind to a promoter It uses one strand as a template to direct synthesis of a complementary sequence of RNA. The length of the bubble is ∼12 to 14 bp The length of RNA-DNA hybrid within it is ∼8 to 9 bp. Figure 11.3

Figure 11.04: The transcription bubble moves along DNA. Ex Biochem c11-transcription Figure 11.04: The transcription bubble moves along DNA.

Figure 11.05: RNA polymerase surrounds the bubble. Ex Biochem c11-transcription Figure 11.05: RNA polymerase surrounds the bubble.

Transcription Reaction Has 3 Stages Ex Biochem c11-transcription Transcription Reaction Has 3 Stages Template recognition: bind to promoter Initiation: RNA polymerase initiates transcription after binding to a promoter site on DNA. Elongation: During elongation the transcription bubble moves along DNA. The RNA chain is extended in the 5′–3′ direction. Termination: When transcription stops: the DNA duplex reforms RNA polymerase dissociates at a terminator site Figure 11.6

11.4 Phage T7 RNA Polymerase Is a Useful Model System Ex Biochem c11-transcription 11.4 Phage T7 RNA Polymerase Is a Useful Model System T3 and T7 phage RNA polymerases are single polypeptides. They have minimal activities in recognizing a small number of phage promoters. Crystal structures of T7 RNA polymerase with DNA identify: the DNA-binding region the active site Figure 11.7

Figure 11.07: T7 RNA polymerase has a single subunit. Ex Biochem c11-transcription Figure 11.07: T7 RNA polymerase has a single subunit.

Figure 11.08: RNA polymerase has a channel for DNA. Ex Biochem c11-transcription Figure 11.08: RNA polymerase has a channel for DNA. Photo courtesy of Seth Darst, Rockefeller University

Figure 11.09: RNA polymerase surrounds DNA. Ex Biochem c11-transcription Figure 11.09: RNA polymerase surrounds DNA.

Figure 11.10: A top view of RNA polymerase II. Ex Biochem c11-transcription Figure 11.10: A top view of RNA polymerase II. Photo courtesy of Roger Kornberg, Stanford University School of Medicine

Figure 11.11: An end view of RNA polymerase II. Ex Biochem c11-transcription Figure 11.11: An end view of RNA polymerase II. Photo courtesy of Roger Kornberg, Stanford University School of Medicine

Ex Biochem c11-transcription

11.5 A Model for Enzyme Movement Is Suggested by the Crystal Structure Ex Biochem c11-transcription 11.5 A Model for Enzyme Movement Is Suggested by the Crystal Structure DNA moves through a groove in yeast RNA polymerase that makes a sharp turn at the active site. Figure 11.12

Figure 11.14: Polymerases must make and break bonds. Ex Biochem c11-transcription Figure 11.14: Polymerases must make and break bonds.

Ex Biochem c11-transcription A protein bridge changes conformation to control the entry of nucleotides to the active site. Figure 11.15

11.6 Bacterial RNA Polymerase Consists of Multiple Subunits Ex Biochem c11-transcription 11.6 Bacterial RNA Polymerase Consists of Multiple Subunits Bacterial RNA core polymerases are ∼500 kD multisubunit complexes with the general structure α2ββ′ RNA polymerase from E. Coli as typical model ~7000 in an cell Complete enzyme (holoenzyme) ~465 kD Figure 11.16

11.10 How Does RNA Polymerase Find Promoter Sequences? Ex Biochem c11-transcription 11.10 How Does RNA Polymerase Find Promoter Sequences? The rate at which RNA polymerase binds to promoters is too fast to be accounted for by random diffusion. Figure 11.22

Ex Biochem c11-transcription RNA polymerase probably: binds to random sites on DNA exchanges them with other sequences very rapidly until a promoter is found Figure 11.23

11.12 Promoter Recognition Depends On Consensus Sequences Ex Biochem c11-transcription 11.12 Promoter Recognition Depends On Consensus Sequences A sequence of DNA whose function is to be recognized by proteins Usually cis-acting A promoter is defined by the presence of short consensus sequences at specific locations. The promoter consensus sequences consist of: a purine at the startpoint the hexamer TATAAT centered at –10 another hexamer centered at –35 Separation between -10 and -35 UP element (sometimes), located further upstream Individual promoters usually differ from the consensus at one or more positions.

Figure 2.16: Proteins bind to cis-acting control sites. Ex Biochem c11-transcription Figure 2.16: Proteins bind to cis-acting control sites.

Figure 2.17: Mutations in control sites are cis-acting. Ex Biochem c11-transcription Figure 2.17: Mutations in control sites are cis-acting.

Figure 11.26: The promoter has three components. Ex Biochem c11-transcription Figure 11.26: The promoter has three components.

11.13 Promoter Efficiencies Can Be Increased or Decreased by Mutation Ex Biochem c11-transcription 11.13 Promoter Efficiencies Can Be Increased or Decreased by Mutation Down mutations: to decrease promoter efficiency usually decrease conformance to the consensus sequences. Up mutations have the opposite effect. Mutations in the –35 sequence usually affect initial binding of RNA polymerase.

Ex Biochem c11-transcription Mutations in the –10 sequence usually affect the melting reaction that converts a closed to an open complex. Figure 11.27

11.20 Bacterial RNA Polymerase Terminates at Discrete Sites Ex Biochem c11-transcription 11.20 Bacterial RNA Polymerase Terminates at Discrete Sites Termination may require both: recognition of the terminator sequence in DNA formation of a hairpin structure in the RNA product Figure 11.45

11.21 There Are Two Types of Terminators in E. coli Ex Biochem c11-transcription 11.21 There Are Two Types of Terminators in E. coli Intrinsic terminators consist of: a G-C-rich hairpin in the RNA product followed by a U-rich region in which termination occurs Rho-dependent terminators Figure 11.46

11.22 How Does Rho Factor Work? Ex Biochem c11-transcription Rho factor is a terminator protein that: binds to a rut site on nascent RNA tracks along the RNA to release it from the RNA–DNA hybrid structure at the RNA polymerase Figure 11.47

Figure 11.48: A rut site has a biased base composition. Ex Biochem c11-transcription Figure 11.48: A rut site has a biased base composition. rich in C, poor in G, no secondary structure

11.23 Antitermination Is a Regulatory Event Ex Biochem c11-transcription 11.23 Antitermination Is a Regulatory Event Termination is prevented when antitermination proteins act on RNA polymerase. This causes it to read through a specific terminator or terminators. Figure 11.51

Ex Biochem c11-transcription Phage lambda has two antitermination proteins, pN and pQ. They act on different transcription units. Figure 11.52

Video sources for transcription Ex Biochem c11-transcription Video sources for transcription http://tw.youtube.com/watch?v=WsofH466lqk http://tw.youtube.com/watch?v=P6Nyce-4oG4 Transcription factors

Ex Biochem c11-transcription http://www.sabiosciences.com/pathway.php?sn=Transcription_of_mRNA