7mkj | pdb_00007mkj
Cryo-EM structure of Escherichia coli RNA polymerase bound to T7A1 promoter DNA
Structural highlights
FunctionRPOA_ECOLI DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. This subunit plays an important role in subunit assembly since its dimerization is the first step in the sequential assembly of subunits to form the holoenzyme.[HAMAP-Rule:MF_00059] Publication Abstract from PubMedThe first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 degrees C: lambdaP(R) (t(1/2) approximately 10 h), T7A1 (t(1/2) approximately 4 min), and a point mutant in lambdaP(R) (lambdaP(R-5C)) (t(1/2) approximately 2 h). Two distinct RPo conformers are populated at lambdaP(R), likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle. Structural origins of Escherichia coli RNA polymerase open promoter complex stability.,Saecker RM, Chen J, Chiu CE, Malone B, Sotiris J, Ebrahim M, Yen LY, Eng ET, Darst SA Proc Natl Acad Sci U S A. 2021 Oct 5;118(40):e2112877118. doi: , 10.1073/pnas.2112877118. PMID:34599106[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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