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 σ⁷⁰-dependent gene transcription through interacting with σ⁷⁰ region 4 and the zinc binding domain (ZBD) of E. coli RNA polymerase (EcoRNAP) β′ 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 β′ subunit (β′ΝΤΗ) and ω subunit, which contributes to stabilize the SspA-EcoRNAP σ⁷⁰ holoenzyme complex. Sequence alignments and phylogenetic tree analyses of N-terminal sequences of β′ subunit from different classes of bacteria revealed that β′ΝΤΗ is highly conserved and exclusively found in low-GC-content Gram-negative bacteria that harbor SspA, implying a co-evolution of β′ΝΤΗ and SspA. The transcription assays of wild-type SspA and its mutants demonstrated the interaction between SspA and β′ΝΤΗ 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.