Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

Bin Liu, Yuhong Zuo, Thomas A. Steitz

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stressresponsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

Original languageEnglish (US)
Pages (from-to)4051-4056
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number15
DOIs
StatePublished - Apr 12 2016

Bibliographical note

Funding Information:
Acknowledgments: We thank the staff of Argonne National Laboratory beamlines 24-ID-C and 24-ID-E for help during data collection, the Center for Structural Biology Facility at Yale University for computational support, and Dr. Jimin Wang for contributions to our data processing and structural refinement. This work was supported by NIH Grant GM22778 (to T.A.S.). T.A.S. is an investigator of the Howard Hughes Medical Institute.

Keywords

  • Promoter recognition
  • Pyrophosphate release
  • RNA polymerase
  • Transcription initiation
  • σ factor

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