Abstract
Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being ‘capped’ at the 5′ end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription in vitro. We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the −1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.
Highlights
IntroductionFor few decades multi-subunit RNA polymerase (RNAP) from Escherichia coli was known to be able to start RNA synthesis with cellular nucleotide coenzymes, adenosine derivatives NAD+ (nicotinamide adenine dinucleotide), NADH (reduced form of NAD+) and FAD (flavin adenine dinucleotide) [1]
For few decades multi-subunit RNA polymerase (RNAP) from Escherichia coli was known to be able to start RNA synthesis with cellular nucleotide coenzymes, adenosine derivatives NAD+, NADH and FAD [1]
Of bacterial RNAs in vivo, another adenine containing cofactor FAD can be incorporated by RNAP in sequencedependent manner, and cap RNAs on +1A (T in the +1 position of the template strand) promoters
Summary
For few decades multi-subunit RNA polymerase (RNAP) from Escherichia coli was known to be able to start RNA synthesis with cellular nucleotide coenzymes, adenosine derivatives NAD+ (nicotinamide adenine dinucleotide), NADH (reduced form of NAD+) and FAD (flavin adenine dinucleotide) [1]. In 2015 those RNAs in E. coli were captured via 5 NAD+ moiety and identified by generation sequencing [3] It transpired that those RNAs were mainly regulatory sRNA and some mRNAs. Only relatively small proportion of the whole population of the particular RNA was NADylated in vivo. Crystal structures of initiation complex containing dinucleotide RNA products (to avoid confusion, here and after, we refer to the RNA length counting NAD+ and other dinucleotide co-factors as a single nucleotide) initiated with adenosine triphosphate (ATP), NAD+ and dpCoA were solved for Thermus thermophilus RNAP [4] demonstrated that, apart from interactions common for all three cofactors, contacts of NAD+ moiety include side chains of  subunits residues D516 and H1237. The authors proposed that nicotinamide moiety of NAD+ may rotate to interact with the −1 position of the template, explaining different efficiencies of NAD+ incorporation on different promoters
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