Role of the RNA polymerase trigger loop in transcript elongation, cleavage, and pausing

Abstract

Multisubunit RNA polymerases (RNAPs) mediate gene expression in all cellular organisms. However, the structural bases of nucleotide addition, of regulatory mechanisms for RNA chain elongation such as transcriptional pausing, and of transcript cleavage by RNAP remain poorly understood. The RNAP active center possesses two distinct types of activities: a nucleotidyl transferase activity that catalyzes nucleotide addition and its reverse reaction, pyrophosphorolysis; and a polynucleotide hydrolytic activity that cleaves the RNA transcript, a reaction that can be accelerated by transcript cleavage factors GreA and GreB. We find that the ability of the trigger loop (a conserved mobile structure located near the secondary channel of RNAP) to fold into two alpha‐helices immediately adjacent to the catalytic center, is required for its nucleotidyl transferase activity (nucleotide addition and pyrophosphorolysis) but not for its polynucleotide hydrolytic activity (intrinsic transcript cleavage and GreB‐stimulated cleavage). In addition, we find that transcriptional pauses, which regulate transcript elongation by temporarily interrupting the nucleotide addition cycle, are accompanied by a catalytic‐center rearrangement that appears to allow fraying of the RNA 3́ nucleotide away from the DNA template and to constrain the trigger‐loop conformation in a way that interferes with nucleotide addition.