Prokaryotic Transcription Chapter 17 Prokaryotic Transcription

Figure 17.0.1: The function of RNA polymerase is to copy one strand of duplex DNA into RNA.

Figure 17.0.2: A transcription unit is a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator.

Figure 17. 3: DNA strands separate to form a transcription bubble Figure 17.0.3: DNA strands separate to form a transcription bubble. RNA is synthesized by complementary base pairing with one of the DNA strands.

Figure 17.0.4: Transcription takes place in a bubble, in which RNA is synthesized by base pairing with one strand of DNA in the transiently unwound region. As the bubble progresses, the DNA duplex reforms behind it, displacing the RNA in the form of a single polynucleotide chain.

Figure 17.0.5: During transcription, the bubble is maintained within bacterial RNA polymerase, which unwinds and rewinds DNA and synthesizes RNA.

Figure 17.0.6: Transcription has three stages: The enzyme binds to the promoter and melts DNA and remains stationary during initiation; moves along the template during elongation; and dissociates at termination.

Figure 17.0.7: Eubacterial RNA polymerases have five types of subunits: α, β, βʹ, and ω have rather constant sizes in different bacterial species, but σ varies more widely.

Figure 17.0.8: The upstream face of the core RNA polymerase, illustrating the “crab claw” shape of the enzyme. The β (cyan) and βʹ (pink) subunits of RNA polymerase have a channel for the DNA template. αI is shown in green and αII in yellow; ω is red. Data from K. M. Geszvain and R. Landick (ed. N. P. Higgins). The Bacterial Chromosome. American Society for Microbiology, 2004.

Figure 17.0.9: The structure of RNA polymerase core enzyme for the bacterium Thermus aquaticus, with the β subunit in blue and the βʹ subunit in green. Structure from Protein Data Bank 1HQM. L. Minakhin, et al., Proc. Natl. Acad. Sci. USA 98 (2001): 892–897.

Figure 17. 10: Core enzyme binds indiscriminately to any DNA Figure 17.0.10: Core enzyme binds indiscriminately to any DNA. Sigma factor reduces the affinity for sequence-independent binding and confers specificity for promoters.

Figure 17.0.11: Proposed mechanisms for how RNA polymerase finds a promoter: (a) sliding, (b) intersegment transfer, (c) intradomain association and dissociation or hopping. Data from C. Bustamante, et al., J. Biol. Chem. 274 (1999): 16665–16668.

Figure 17.0.12: RNA polymerase passes through several steps prior to elongation. A closed binary complex is converted to an open form and then into a ternary complex. Data from S. P. Haugen, W. Ross, and R. L. Gourse, Nat. Rev. Microbiol. 6 (2008): 507–519.

Figure 17.0.13: RNA polymerase initially contacts the region from −55 to +20. When sigma dissociates, the core enzyme contracts to −30; when the enzyme moves a few base pairs, it becomes more compactly organized into the general elongation complex.

Figure 17.0.14: DNA elements and RNA polymerase modules that contribute to promoter recognition by sigma factor. Data from S. P. Haugen, W. Ross, and R. L. Gourse, Nat. Rev. Microbiol. 6 (2008): 507–519.

Table 17.0.1: E. coli sigma factors recognize promoters with different consensus sequences.

Figure 17.0.15: The structure of sigma factor in the context of the holoenzyme: −10 and −35 interactions. Sigma factor is extended and its domains are connected by flexible linkers. Illustration adapted from D. G. Vassylyev, et al., Nature 417 (2002): 712–719. Structure from Protein Data Bank 1IW7.

Figure 17. 16: Amino acids in the 2 Figure 17.0.16: Amino acids in the 2.4 α-helix of β70 contact specific bases in the coding strand of the −10 promoter sequence.

Figure 17.0.17: The N-terminus of sigma blocks the DNA-binding regions from binding to DNA. When an open complex forms, the N-terminus swings 20 Å away, and the two DNA-binding regions separate by 15 Å.

Figure 17.0.18: Sigma factor has an elongated structure that extends along the surface of the core subunits when the holoenzyme is formed.

Figure 17.0.19: DNA initially contacts sigma factor (pink) and core enzyme (gray). It moves deeper into the core enzyme to make contacts at the −10 sequence. When sigma is released, the width of the passage containing DNA increases. Reprinted by permission from Macmillan Publishers Ltd: Nature, D. G. Vassylyev, et al., vol. 417, pp. 712–719, copyright 2002. Photo courtesy of Shigeyuki Yokoyama, The University of Tokyo.

Figure 17.0.20: Footprinting identifies DNA-binding sites for proteins by their protection against nicking.

Figure 17.0.21: One face of the promoter contains the contact points for RNA.

Figure 17.0.22: Sequence-specific recognition of the −10 element by region 2 of σ. The DNA backbone is represented by green circles, bases of the nontemplate strand by dark blue polygons, and bases of the template strand by light blue polygons. The sequence of the nontemplate strand corresponds to the consensus of the −10 element. Region 2 of σ is shown as an orange polygon. Data from X. Liu, et al., Cell 147 (2011): 1218–1219.

Figure 17.0.23: Sigma factor and core enzyme recycle at different points in transcription.

Figure 17.0.24: The A model showing the structure of RNA polymerase through the main channel. Subunits are color-coded as follows: βʹ, pink; β, cyan; αI, green; αII, yellow; ω, red. Data from K. M. Geszvain and R. Landick (ed. N. P. Higgins). The Bacterial Chromosome. American Society for Microbiology, 2004.

Figure 17.0.25: DNA is forced to make a turn at the active site by a wall of protein. Nucleotides may enter the active site through a pore in the protein.

Figure 17.0.26: Movement of a nucleic acid polymerase requires breaking and remaking bonds to the nucleotides at fixed positions relative to the enzyme structure. The nucleotides in these positions change each time the enzyme moves a base along the template.

Figure 17.0.27: The DNA sequences required for termination are located upstream of the terminator sequence. Formation of a hairpin in the RNA may be necessary.

Figure 17.0.28: Intrinsic terminators include palindromic regions that form hairpins varying in length from 7 to 20 bp. The stem-loop structure includes a G-C–rich region and is followed by a run of U residues.

Figure 17.0.29: Rho factor binds to RNA at a rut site and translocates along RNA until it reaches the RNA–DNA hybrid in RNA polymerase, where it releases the RNA from the DNA.

Figure 17.0.30: A rut site has a sequence rich in C and poor in G preceding the actual site(s) of termination. The sequence corresponds to the 3ʹ end of the RNA.

Figure 17.0.31: Rho has an N-terminal, RNA-binding domain and a C-terminal ATPase domain. A hexamer in the form of a gapped ring binds RNA along the exterior of the N-terminal domains. The 5ʹ end of the RNA is bound by a secondary binding site in the interior of the hexamer.

Figure 17.0.32: The action of rho factor may create a link between transcription and translation when a rho-dependent terminator lies soon after a nonsense mutation.

Figure 17.0.33: Transcription generates more tightly wound (positively supercoiled) DNA ahead of RNA polymerase, while the DNA behind becomes less tightly wound (negatively supercoiled).

Figure 17.0.34: T7 RNA polymerase has a specificity loop that binds positions −7 to −11 of the promoter while positions −1 to −4 enter the active site.

Figure 17.0.35: The sigma factor associated with core enzyme determines the set of promoters at which transcription is initiated.

Table 17. 2: In addition to σ70, E Table 17.0.2: In addition to σ70, E. coli has several sigma factors that are induced by particular environmental conditions. (A number in the name of a factor indicates its mass.)