Abstract
Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor–although shorter and unrelated in sequence–follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent transcription initiation.
Highlights
Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 and region 4
Structures were determined using recombinant Mycobacterium tuberculosis (Mtb) RNA polymerase (RNAP) core enzyme prepared by co-expression of Mtb RNAP subunit genes in E. coli, recombinant Mtb σL, and synthetic nucleic-acid scaffolds based on the sequence of the σL-dependent promoter P-sigL36–38 (Supplementary Figs. 1, 2)
Our results regarding the connector between σ factors: region 2 (σR2) and σR4 of an ECF σ factor, in conjunction with previous results, indicate that all classes of bacterial σ factors contain structural modules that enter the RNAP active-center cleft to interact with templatestrand ssDNA and leave the RNAP active-center cleft by threading through the RNAP RNA-exit channel, providing mechanisms to facilitate de novo initiation, to coordinate extension of the nascent RNA with abortive initiation and initialtranscription pausing, and to coordinate entry of RNA into the RNA-exit channel with promoter escape
Summary
Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). We report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. No structural information previously has been reported for RNAP holoenzymes or transcription initiation complexes containing ECF σ factors. In the absence of structural information for ECF σ factors, it has been unclear how ECF σ factors, despite lacking sequences homologous to the σR3/4 linker of group-1 σ factors, are able to connect σR2 and σR4 with an appropriate spacing to recognize promoter -10 and -35 elements, are able to pre-organize the DNA template strand to facilitate initiatingnucleotide binding and de novo transcription initiation; and are able to coordinate entry of RNA into the RNA-exit channel with promoter escape. In the absence of structural information, and with comparatively limited sequence similarity between σR2 of ECF σ factors and σR2 of group-1 σ factors[1], it has been unclear whether σR2 of ECF σ factors adopts the same fold as σR2 of group-1 σ factors and uses the same strategy to bind and unwind the promoter -10 element as group-1 σ factors
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