Global regulation via modulation of ribosome pausing by the ABC-F protein EttA.

Having multiple rounds of translation of the same mRNA creates dynamic complexities along with opportunities for regulation related to ribosome pausing and stalling at specific sequences. Yet, mechanisms controlling these critical processes and the principles guiding their evolution remain poorly understood. Through genetic, genomic, physiological, and biochemical approaches, we demonstrate that regulating ribosome pausing at specific amino acid sequences can produce ~2-fold changes in protein expression levels which strongly influence cell growth and therefore evolutionary fitness.

Regulation of the macrolide resistance ABC-F translation factor MsrD.

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors that provide resistance against clinically important ribosome-targeting antibiotics which are proliferating among pathogens. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure. We show that binding of cladinose-containing macrolides to the ribosome prompts insertion of the leader peptide MsrDL into a crevice of the ribosomal exit tunnel, which is conserved throughout bacteria and eukaryotes.

Processing and decay of 6S-1 and 6S-2 RNAs in .

Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1.

Dynamic Membrane Localization of RNase Y in Bacillus subtilis.

Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells.

In Vitro Study of the Major Bacillus subtilis Ribonucleases Y and J.

The metabolic instability of mRNA is fundamental to the adaptation of gene expression. In bacteria, mRNA decay follows first-order kinetics and is primarily controlled at the steps initiating degradation. In the model Gram-positive organism Bacillus subtilis, the major mRNA decay pathway initiates with an endonucleolytic cleavage by the membrane-associated RNase Y.

YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity.

Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E.

Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells.

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3'-phosphoadenosine 5'-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn.

Proteome analysis of the phenotypic variation process in Photorhabdus luminescens.

Photorhabdus luminescens is an insect pathogen associated with specific soil nematodes. The bacterium has a complex life cycle with a symbiotic stage in which bacteria colonize the intestinal tract of the nematodes, and a pathogenic stage against susceptible larval-stage insect. Symbiosis-"deficient" phenotypic variants (known as secondary forms) arise during prolonged incubation.

AstR-AstS, a new two-component signal transduction system, mediates swarming, adaptation to stationary phase and phenotypic variation in Photorhabdus luminescens.

Photorhabdus luminescens is an insect-pathogenic bacterium that forms a symbiosis with specific entomopathogenic nematodes. In this bacterium, a symbiosis-'deficient' phenotypic variant (known as the secondary variant or form II) arises at a low frequency during prolonged incubation. A knock-out mutant was generated of the regulator of a newly identified two-component regulatory system, designated AstR-AstS.