Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

Abstract

<jats:p>DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, <jats:italic>in vitro</jats:italic> transcription, kinetic analyses, and <jats:italic>in vivo</jats:italic> cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by <jats:italic>Escherichia coli</jats:italic> RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic <jats:italic>in vivo</jats:italic>, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.</jats:p>

Suppressor mutations in Escherichia coli RNA polymerase alter transcription initiation but do not affect translesion RNA synthesis in vitro

“…The kinetics of RNA extension was strongly delayed in the case of all three lesions. In agreement with published data (50)(51)(52)(53), on the APsite template, RNAP was stalled at two positions, one nucleotide upstream and directly opposite of the lesion, and further RNA synthesis was significantly delayed (Fig. 7A).…”

“…The addition of both factors only slightly inhibited RNA extension on the AP-site template by the WT RNAP (Fig. S8), in agreement with published data showing weak effects of DksA/ppGpp on translesion synthesis by WT E. coli RNAP (53). Similarly, DksA and ppGpp had only minor effect on RNA extension opposite the AP-site by all mutant RNAPs (Fig.…”

“…εA strongly inhibited nucleotide incorporation opposite the lesion, with the major pause observed upstream of the lesion (Fig. 7C) (50,51,53). Remarkably, none of the mutations led to dramatic changes in the kinetics of RNA synthesis and the pattern of extended RNA products on both control and damaged templates.…”

Mechanistic insights into RNA cleavage by bacterial RNA polymerase from a comprehensive mutational screen

“…Notably, in the majority of gram-negative bacteria, the TL contains a large insertion known as SI3 ( Figure 4A ). This addition affects the dynamics of TL to TH transition, with SI3 inhibiting TL folding into TH in certain sequence contexts (Bao & Landick, 2021; Bao et al , 2024), but having little effect in others (Agapov et al , 2020).…”