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Depletion of pre-16S rRNA in starved Escherichia coli cells.

ABSTRACT: Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth.

SUBMITTER: Cangelosi GA

PROVIDER: S-EPMC179279 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Depletion of pre-16S rRNA in starved Escherichia coli cells.

Cangelosi G A GA Brabant W H WH

Journal of bacteriology 19970701 14

Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes ...[more]

PMID: 9226253

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Project description:YbeY is part of a core set of RNases in Escherichia coli and other bacteria. This highly conserved endoribonuclease has been implicated in several important processes such as 16S rRNA 3' end maturation, 70S ribosome quality control, and regulation of mRNAs and small noncoding RNAs, thereby affecting cellular viability, stress tolerance, and pathogenic and symbiotic behavior of bacteria. Thus, YbeY likely interacts with numerous protein or RNA partners that are involved in various aspects of cellular physiology. Using a bacterial two-hybrid system, we identified several proteins that interact with YbeY, including ribosomal protein S11, the ribosome-associated GTPases Era and Der, YbeZ, and SpoT. In particular, the interaction of YbeY with S11 and Era provides insight into YbeY's involvement in the 16S rRNA maturation process. The three-way association between YbeY, S11, and Era suggests that YbeY is recruited to the ribosome where it could cleave the 17S rRNA precursor endonucleolytically at or near the 3' end maturation site. Analysis of YbeY missense mutants shows that a highly conserved beta-sheet in YbeY-and not amino acids known to be important for YbeY's RNase activity-functions as the interface between YbeY and S11. This protein-interacting interface of YbeY is needed for correct rRNA maturation and stress regulation, as missense mutants show significant phenotypic defects. Additionally, structure-based in silico prediction of putative interactions between YbeY and the Era-30S complex through protein docking agrees well with the in vivo results. IMPORTANCE:Ribosomes are ribonucleoprotein complexes responsible for a key cellular function, protein synthesis. Their assembly is a highly coordinated process of RNA cleavage, RNA posttranscriptional modification, RNA conformational changes, and protein-binding events. Many open questions remain after almost 5 decades of study, including which RNase is responsible for final processing of the 16S rRNA 3' end. The highly conserved RNase YbeY, belonging to a core set of RNases essential in many bacteria, was previously shown to participate in 16S rRNA processing and ribosome quality control. However, detailed mechanistic insight into YbeY's ribosome-associated function has remained elusive. This work provides the first evidence that YbeY is recruited to the ribosome through interaction with proteins involved in ribosome biogenesis (i.e., ribosomal protein S11, Era). In addition, we identified key residues of YbeY involved in the interaction with S11 and propose a possible binding mode of YbeY to the ribosome using in silico docking.

Dataset Information

Depletion of pre-16S rRNA in starved Escherichia coli cells.

Publications

Depletion of pre-16S rRNA in starved Escherichia coli cells.

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