Sinorhizobium meliloti YbeY is a zinc-dependent single-strand specific endoribonuclease that plays an important role in 16S ribosomal RNA processing

Abstract Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3′ end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.

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Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli

Journal of Molecular Biology ◽

10.1016/j.jmb.2018.04.009 ◽

2018 ◽

Vol 430 (12) ◽

pp. 1745-1759 ◽

Cited By ~ 16

Author(s):

Brian A. Smith ◽

Neha Gupta ◽

Kevin Denny ◽

Gloria M. Culver

Keyword(s):

16S Rrna ◽

Rrna Processing ◽

Subunit Assembly ◽

E Coli ◽

30S Subunit

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Subunit Composition of Ribosome in the yqgF Mutant Is Deficient in pre-16S rRNA Processing of Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000494494 ◽

2018 ◽

Vol 28 (4) ◽

pp. 179-182

Author(s):

Tatsuaki Kurata ◽

Shinobu Nakanishi ◽

Masayuki Hashimoto ◽

Masato Taoka ◽

Toshiaki Isobe ◽

...

Keyword(s):

Escherichia Coli ◽

16S Rrna ◽

Active Form ◽

Rrna Processing ◽

Rnase E ◽

Rnase Iii ◽

Primary Transcript ◽

E Coli ◽

Copy Numbers ◽

Inactive Form

Escherichia coli 16S, 23S, and 5S ribosomal RNAs (rRNAs) are transcribed as a single primary transcript, which is subsequently processed into mature rRNAs by several RNases. Three RNases (RNase III, RNase E, and RNase G) were reported to function in processing the 5′-leader of precursor 16S rRNA (pre-16S rRNA). Previously, we showed that a novel essential YqgF is involved in that processing. Here we investigated the ribosome subunits of the yqgFts mutant by LC-MS/MS. The mutant ribosome had decreased copy numbers of ribosome protein S1, suggesting that the yqgF gene enables incorporation of ribosomal protein S1 into ribosome by processing of the 5′-end of pre-16S rRNA. The ribosome protein S1 is essential for translation in E. coli; therefore, our results suggest that YqgF converts the inactive form of newly synthesized ribosome into the active form at the final step of ribosome assembly.

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Orthogonal translation enables heterologous ribosome engineering in E. coli

Nature Communications ◽

10.1038/s41467-020-20759-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Natalie S. Kolber ◽

Ranan Fattal ◽

Sinisa Bratulic ◽

Gavriela D. Carver ◽

Ahmed H. Badran

Keyword(s):

Synthetic Biology ◽

16S Rrna ◽

General Framework ◽

Living Cells ◽

Rrna Processing ◽

Ribosome Engineering ◽

Subunit Exchange ◽

E Coli ◽

Ribosome Binding ◽

Promising Avenue

AbstractThe ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.

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RNase AM, a 5′ to 3′ exonuclease, matures the 5′ end of all three ribosomal RNAs in E. coli

Nucleic Acids Research ◽

10.1093/nar/gkaa260 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5616-5623 ◽

Cited By ~ 2

Author(s):

Chaitanya Jain

Keyword(s):

Escherichia Coli ◽

16S Rrna ◽

Rna Metabolism ◽

23S Rrna ◽

Rrna Processing ◽

Ribosomal Rnas ◽

E Coli ◽

Processing Step ◽

Rnase G ◽

Prior Processing

Abstract Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5′-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5′ to 3′ exonuclease, performs the last step of 5S rRNA 5′-end maturation. RNase AM was also found to generate the mature 5′ end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5′ end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5′ to 3′ RNA exonuclease identified in E. coli.

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Elevated Levels of Era GTPase Improve Growth, 16S rRNA Processing, and 70S Ribosome Assembly ofEscherichia coliLacking Highly Conserved Multifunctional YbeY Endoribonuclease

Journal of Bacteriology ◽

10.1128/jb.00278-18 ◽

2018 ◽

Vol 200 (17) ◽

Cited By ~ 11

Author(s):

Anubrata Ghosal ◽

Vignesh M. P. Babu ◽

Graham C. Walker

Keyword(s):

16S Rrna ◽

Ribosome Biogenesis ◽

Growth Defect ◽

Ribosome Assembly ◽

Rrna Processing ◽

Content Type ◽

E Coli ◽

New Class ◽

70S Ribosome

ABSTRACTYbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era inEscherichia colipartially suppresses the growth defect of a ΔybeYstrain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression ofVibrio choleraeEra similarly partially suppresses the defects of anE. coliΔybeYstrain, indicating that this property of Era is conserved in bacteria other thanE. coli.IMPORTANCEThis work provides insight into the critical, but still incompletely understood, mechanism of processing of theE. coli16S rRNA 3′ terminus. The highly conserved GTPase Era is known to bind to the precursor of the 16S rRNA near its 3′ end. Both the endoribonuclease YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3′-end processing. The results reported here offer additional insights into the role of Era in 16S rRNA 3′-end maturation and into the relationship between the action of the endoribonuclease YbeY and that of the four exoribonucleases. This study also hints at why YbeY is essential only in some bacteria and suggests that YbeY could be a target for a new class of antibiotics in these bacteria.

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Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128833 ◽

1987 ◽

Vol 45 ◽

pp. 910-911

Author(s):

M. Boublik ◽

V. Mandiyan ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Compact Structure ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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The Location of Substitutions and Bacterial Genome Arrangements

Genome Biology and Evolution ◽

10.1093/gbe/evaa260 ◽

2020 ◽

Author(s):

Daniella F Lato ◽

G Brian Golding

Keyword(s):

Sinorhizobium Meliloti ◽

Bacterial Genome ◽

Evolutionary Analysis ◽

Origin Of Replication ◽

Ancestral Reconstruction ◽

Molecular Change ◽

E Coli ◽

A Genome ◽

The Impact ◽

Molecular Evolutionary Analysis

Abstract Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, while substitution rates usually increases with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and distance from the origin of replication is often significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), while others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic.

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Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies

Biochemical Journal ◽

10.1042/bj20060660 ◽

2006 ◽

Vol 400 (1) ◽

pp. 105-114 ◽

Cited By ~ 27

Author(s):

Eric Di Luccio ◽

Robert A. Elling ◽

David K. Wilson

Keyword(s):

Escherichia Coli ◽

Sinorhizobium Meliloti ◽

Thermotoga Maritima ◽

Xylose Reductase ◽

Sulfolobus Solfataricus ◽

Sequence Comparisons ◽

E Coli ◽

Fluorescence Measurements ◽

Dual Specificity ◽

Substrate Dependence

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2′-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.

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