A unique binding between SspA and RNAP β′NTH across low-GC Gram-negative bacteria facilitates SspA-mediated transcription regulation

Stringent starvation protein A (SspA) involved in nucleotide metabolism, acid tolerance and virulence of bacteria has been demonstrated to function as a transcription factor to regulate sigma(70)-dependent gene transcription through interacting with sigma(70) region 4 and the zinc binding domain (ZBD) of E. coli. RNA polymerase (EcoRNAP) beta' subunit simultaneously. Despite extensive biochemical and structural analyses were reported recently, the interactions of SspA with RNAP are not comprehensively understood. Here, we reprocessed our previous cryo-EM dataset of EcoRNAP-promoter open complex with SspA (SspA-RPo) and obtained a significantly improved density map. Unexpectedly, the new map showed that SspA interacts with both N-terminal helix of beta' subunit (beta'NTH) and (i) subunit, which contributes to stabilize the SspA-EcoRNAP sigma(70) holoenzyme complex. Sequence alignments and phylogenetic tree analyses of N-terminal sequences of beta' subunit from different classes of bacteria revealed that beta'NTH is highly conserved and exclusively found in low-GC-content Gram-negative bacteria that harbor SspA, implying a co-evolution of beta'NTH and SspA. The transcription assays of wild-type SspA and its mutants demonstrated the interaction between SspA and beta'NTH facilitates the transcription regulation of SspA. Together, our results provide a more comprehensive insight into the interactions between SspA and RNAP and their roles in bacterial transcription regulation. (C) 2021 Elsevier Inc. All rights reserved.

Automated summary

Stringent starvation protein A (SspA) functions as a transcription factor in bacteria, interacting with specific subunits of RNA polymerase to regulate gene transcription. The interaction of SspA with the N-terminal helix of the beta' subunit and (i) subunit helps stabilize the SspA-RNA polymerase complex. Furthermore, sequence comparisons suggest a co-evolution of these interactions in specific low-GC Gram-negative bacteria.