Biology subjects

Innis, C. A.

Publications and source records attributed to Innis, C. A..

4 recordsLinked to original sources

bioRxiv2022

Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs

With antibiotic-resistant bacteria threatening our ability to treat common infections, new lead compounds with distinct target binding sites and limited cross-resistance are urgently needed. Natural products that inhibit the bacterial ribosome - a target for more than half of the antibiotics in use today - are a promising source of such leads and have the potential to be developed into potent drugs through structure-guided design. However, because the mechanisms of action of many of these compounds are not well understood, they are often poor candidates for a structure-based approach. Here, we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X (TcmX) primarily inhibits the formation of a peptide bond between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3 adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of TcmX on the bacterial ribosome and suggests a path forward for the development of novel antibiotics based on a common aromatic polyketide scaffold.

molecular biology↗

bioRxiv2021

Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure and also demonstrate that MsrD twin-ATPase sites work asymmetrically to mediate the dynamic of MsrD interaction with the ribosome. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into an undocumented conserved crevice of the ribosomal exit tunnel concomitantly with 23S rRNA rearrangements that prevent peptide bond formation and preclude accommodation of release factors. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

microbiology↗

bioRxiv2021

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

molecular biology↗

bioRxiv2019

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1-3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in {gamma}-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

microbiology↗