Biology subjects

Remme, J.

Publications and source records attributed to Remme, J..

3 recordsLinked to original sources

bioRxiv2026

23S rRNA modifications stimulate catalytic activity and prevent the formation of alternative structures

Ribosomal RNA modifications cluster around the peptidyl transferase center (PTC), the catalytic center of the ribosome, yet their collective functional roles remain unclear. Here we analyze Escherichia coli ribosomes lacking 11 or 12 modifications near the PTC. Using kinetic assays, we show these hypo-modified ribosomes catalyze peptide bond formation at rates two- to threefold lower than wild-type and exhibit reduced thermal stability. Cryo-electron microscopy of hypo-modified ribosomes reveals multiple alternative conformations of the PTC and exit tunnel regions, disrupting native stacking and hydrogen bonding critical for positioning of tRNA substrates. These findings indicate that rRNA modifications stabilize the native PTC structure, preventing formation of alternative, nonfunctional conformations and thereby enhance catalytic efficiency. Our study provides insight into how rRNA modifications fine-tune ribosome function by maintaining structural integrity essential for efficient translation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/708810v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f06f16org.highwire.dtl.DTLVardef@d189aorg.highwire.dtl.DTLVardef@1ca55acorg.highwire.dtl.DTLVardef@bec055_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally-important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the "critical region" of the peptidyl transferase center (PTC) in 23S rRNA that exhibited only a minor growth defect at 37{degrees}C (although major at 20{degrees}C). However, our combined KO of modification enzymes RluC and RlmE resulted in conditional lethality (at 20{degrees}C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20 and 37{degrees}C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Rates of protein synthesis in vivo, as judged by the kinetics of {beta}-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37{degrees}C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

biochemistry↗

bioRxiv2022

A conundrum of r-protein stability: unbalanced stoichiometry of r-proteins during stationary phase in Escherichia coli

Bacterial ribosomes are composed of three ribosomal RNA (rRNA) and over 50 ribosomal protein (r-protein) molecules. R-proteins are essential for ribosome assembly, structural stability, and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during the exponential and early stationary growth phases. Ribosomes are degraded in stationary phase, however, the stability and fate of r-proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during the extended stationary phase in E.coli. We show that: (a) the quantity of ribosomes per cell mass decreases in the stationary phase. (b) 70S ribosomes contain r-proteins in stoichiometric amounts. (c) 30S subunits are degraded faster than 50S. (d) In total proteome, the quantity of 21 r-proteins decreases during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA. (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus r-proteins are present in non-stochiometric amounts in the proteome of E. coli during the extended stationary phase. IMPORTANCERibosome degradation has been extensively described from the viewpoint of its main component - rRNA. Here we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability in the ribosomes as well as in the whole-cell proteome during stationary phase in E. coli. R-proteins are considered to be very stable in the proteome. Here we show that a specific set of r-proteins are rapidly degraded after release from the rRNA. The degradation of r-proteins is an intriguing new aspect of r-protein metabolism in bacteria.

molecular biology↗