scholarly journals Arginine Methylation Regulates Ribosome CAR Function

2021 ◽  
Vol 22 (3) ◽  
pp. 1335
Author(s):  
Kristen Scopino ◽  
Carol Dalgarno ◽  
Clara Nachmanoff ◽  
Daniel Krizanc ◽  
Kelly M. Thayer ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Arginine Methylation ◽  
Translation Regulation ◽  
E Coli ◽  
Pi Stacking ◽  
Cellular Context ◽  
Guanidinium Group ◽  
Stressed Cells ◽  
A Site ◽  
Interaction Surface

The ribosome CAR interaction surface is hypothesized to provide a layer of translation regulation through hydrogen-bonding to the +1 mRNA codon that is next to enter the ribosome A site during translocation. The CAR surface consists of three residues, 16S/18S rRNA C1054, A1196 (E. coli 16S numbering), and R146 of yeast ribosomal protein Rps3. R146 can be methylated by the Sfm1 methyltransferase which is downregulated in stressed cells. Through molecular dynamics analysis, we show here that methylation of R146 compromises the integrity of CAR by reducing the cation-pi stacking of the R146 guanidinium group with A1196, leading to reduced CAR hydrogen-bonding with the +1 codon. We propose that ribosomes assembled under stressed conditions have unmethylated R146, resulting in elevated CAR/+1 codon interactions, which tunes translation levels in response to the altered cellular context.

scholarly journals Arginine Methylation Regulates Ribosome CAR Function

2020 ◽  
Author(s):  
Kristen Scopino ◽  
Carol Dalgarno ◽  
Clara Nachmanoff ◽  
Daniel Krizanc ◽  
Kelly M. Thayer ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Arginine Methylation ◽  
Translation Regulation ◽  
E Coli ◽  
Pi Stacking ◽  
Cellular Context ◽  
Guanidinium Group ◽  
Stressed Cells ◽  
A Site ◽  
Interaction Surface

AbstractThe ribosome CAR interaction surface is hypothesized to provide a layer of translation regulation through hydrogen-bonding to the +1 mRNA codon that is next to enter the ribosome A site during translocation. The CAR surface consists of three residues, 16S/18S rRNA C1054, A1196 (E. coli 16S numbering), and R146 of yeast ribosomal protein Rps3. R146 can be methylated by the Sfm1 methyltransferase which is downregulated in stressed cells. Through molecular dynamics analysis, we show here that methylation of R146 compromises the integrity of CAR by reducing the pi stacking of the R146 guanidinium group with A1196, leading to reduced CAR hydrogen-bonding with the +1 codon. We propose that ribosomes assembled under stressed conditions have unmethylated R146, resulting in elevated CAR/+1 codon interactions, which tunes translation levels in response to the altered cellular context.

scholarly journals A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

2020 ◽  
Author(s):  
Kristen Scopino ◽  
Elliot Williams ◽  
Abdelrahman Elsayed ◽  
William A. Barr ◽  
Daniel Krizanc ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Bonding ◽  
18S Rrna ◽  
Protein Translation ◽  
Base Stacking ◽  
Codon Positions ◽  
Guanidinium Group ◽  
Dynamics Simulations ◽  
A Site ◽  
Interaction Surface

ABSTRACTGCN codons are over-represented in initial codons of ORFs of prokaryote and eukaryote mRNAs. We describe a ribosome rRNA-protein surface that interacts with an mRNA GCN codon when next-in-line for the ribosome A site. The interaction surface is comprised of the edges of two stacked rRNA bases: the Watson-Crick edge of 16S/18S rRNA C1054 and adjacent Hoogsteen edge of A1196 (Escherichia coli 16S rRNA numbering). Also part of the interaction surface, the planar guanidinium group of a conserved Arginine (R146 of yeast ribosomal protein Rps3) is stacked adjacent to A1196. On its other side, the interaction surface is anchored to the ribosome A site through base stacking of C1054 with the wobble anticodon base of the A-site tRNA. Using Molecular Dynamics simulations of a 495-residue subsystem of translocating ribosomes, we observe base pairing of C1054 to nucleotide G at position 1 of the next-in-line codon, consistent with previous cryo-EM observations, and hydrogen bonding of A1196 and R146 to C at position 2. Hydrogen bonding to both of these codon positions is significantly weakened when C at position 2 is changed to G, A or U. These sequence-sensitive mRNA-ribosome interactions at the C1054-A1196-R146 (CAR) surface potentially contribute to GCN-mediated regulation of protein translation.

scholarly journals A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 849 ◽  
Author(s):  
Kristen Scopino ◽  
Elliot Williams ◽  
Abdelrahman Elsayed ◽  
William A. Barr ◽  
Daniel Krizanc ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Protein Translation ◽  
Open Reading Frames ◽  
Base Stacking ◽  
Codon Positions ◽  
Guanidinium Group ◽  
Dynamics Simulations ◽  
A Site ◽  
Interaction Surface ◽  
Reading Frames

A longstanding challenge is to understand how ribosomes parse mRNA open reading frames (ORFs). Significantly, GCN codons are over-represented in the initial codons of ORFs of prokaryote and eukaryote mRNAs. We describe a ribosome rRNA-protein surface that interacts with an mRNA GCN codon when next in line for the ribosome A-site. The interaction surface is comprised of the edges of two stacked rRNA bases: the Watson–Crick edge of 16S/18S rRNA C1054 and the adjacent Hoogsteen edge of A1196 (Escherichia coli 16S rRNA numbering). Also part of the interaction surface, the planar guanidinium group of a conserved Arginine (R146 of yeast ribosomal protein Rps3) is stacked adjacent to A1196. On its other side, the interaction surface is anchored to the ribosome A-site through base stacking of C1054 with the wobble anticodon base of the A-site tRNA. Using molecular dynamics simulations of a 495-residue subsystem of translocating ribosomes, we observed base pairing of C1054 to nucleotide G at position 1 of the next-in-line codon, consistent with previous cryo-EM observations, and hydrogen bonding of A1196 and R146 to C at position 2. Hydrogen bonding to both of these codon positions is significantly weakened when C at position 2 is changed to G, A or U. These sequence-sensitive mRNA-ribosome interactions at the C1054-A1196-R146 (CAR) surface potentially contribute to the GCN-mediated regulation of protein translation.

scholarly journals GCN sensitive protein translation in yeast

2020 ◽  
Author(s):  
William A. Barr ◽  
Ruchi B. Sheth ◽  
Jack Kwon ◽  
Jungwoo Cho ◽  
Jacob W. Glickman ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translation ◽  
Open Reading Frames ◽  
Translation Efficiency ◽  
E Coli ◽  
Positive Effects ◽  
Braking System ◽  
Expression Studies ◽  
A Site ◽  
Interaction Surface

AbstractLevels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons--the ramp region--of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

2022 ◽  
Vol 0 (0) ◽  
Author(s):  
V. Janett Olzog ◽  
Lena I. Freist ◽  
Robin Goldmann ◽  
Jörg Fallmann ◽  
Christina E. Weinberg
Keyword(s):  
Rna Seq ◽  
Site Specific ◽  
Pyrococcus Horikoshii ◽  
Total Rna ◽  
E Coli ◽  
Catalytic Rnas ◽  
Adapter Ligation ◽  
Domains Of Life ◽  
Cyclic Phosphate ◽  
A Site

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals Waterborne illness and injury: a feasibility study of a site-specific predictive model for beach water quality at Beachway Park in the City of Burlington

2021 ◽  
Author(s):  
Ramien Sereshk
Keyword(s):  
Water Quality ◽  
Predictive Model ◽  
Real Time ◽  
Site Specific ◽  
E Coli ◽  
Time Basis ◽  
A Site ◽  
Persistence Model ◽  
Beach Water Quality ◽  
Beach Water

It is commonly assumed that the persistence model, using day-old monitoring results, will provide accurate estimates of real-time bacteriological concentrations in beach water. However, the persistence model frequently provides incorrect results. This study: 1. develops a site-specific predictive model, based on factors significantly influencing water quality at Beachway Park; 2. determines the feasibility of the site-specific predictive model for use in accurately predicting near real-time E. coli levels. A site-specific predictive model, developed for Beachway Park, was evaluated and the results were compared to the persistence model. This critical performance evaluation helped to identify the inherent inaccuracy of the persistence model for Beachway Park, which renders it an unacceptable approach for safeguarding public health from recreational water-borne illnesses. The persistence model, supplemented with a site-specific predictive model, is recommended as a feasible method to accurately predict bacterial levels in water on a near real-time basis.

Bioinspired catalysis and bromoperoxidase like activity of a multistimuli-responsive supramolecular metallogel: supramolecular assembly triggered by pi–pi stacking and hydrogen bonding interactions

2020 ◽  
Vol 44 (14) ◽  
pp. 5410-5418 ◽  
Author(s):  
Sunshine Dominic Kurbah ◽  
Ram A. Lal
Keyword(s):  
Hydrogen Bonding ◽  
Supramolecular Assembly ◽  
Synthesis And Characterization ◽  
Pi Stacking ◽  
Hydrogen Bonding Interactions ◽  
Bromination Reaction ◽  
Oxidative Bromination ◽  
Self Assembled

We report the synthesis and characterization of a new self-assembled VO2-L metallogel. Multi-responsive properties of the gel were also studied and can be used for sensing OH− anions. Bromoperoxidase-like activity of VO2-L metallogel for oxidative bromination reaction was also reported.

Inactivation of Stressed Escherichia coli O157:H7 Cells on the Surfaces of Rocket Salad Leaves by Chlorine and Peroxyacetic Acid

2014 ◽  
Vol 77 (1) ◽  
pp. 32-39 ◽  
Author(s):  
ANAS A. AL-NABULSI ◽  
TAREQ M. OSAILI ◽  
HEBA M. OBAIDAT ◽  
REYAD R. SHAKER ◽  
SADDAM S. AWAISHEH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Deionized Water ◽  
Escherichia Coli O157 ◽  
Apparent Difference ◽  
Peroxyacetic Acid ◽  
Surface Disinfection ◽  
E Coli ◽  
Leafy Greens ◽  
Foodborne Outbreaks ◽  
Stressed Cells

Because Escherichia coli O157:H7 has been frequently associated with many foodborne outbreaks caused by consumption of leafy greens (lettuce, spinach, and celery), this study investigated the ability of deionized water, chlorine, and peroxyacetic acid to detach or inactivate stressed and unstressed cells of E. coli O157:H7 contaminating the surfaces of rocket salad leaves. E. coli O157:H7 cells stressed by acid, cold, starvation, or NaCl exposure, as well as unstressed cells, were inoculated on the surfaces of rocket salad leaves at 4°C. The effectiveness of two sanitizers (200 ppm of chlorine and 80 ppm of peroxyacetic acid) and deionized water for decontaminating the leaves treated with stressed and unstressed E. coli O157:H7 were evaluated during storage at 10 or 25°C for 0.5, 1, 3, and 7 days. It was found that washing with 80 ppm of peroxyacetic acid was more effective and reduced unstressed and stressed cells of E. coli O157:H7 by about 1 log CFU per leaf on the leaves. There was no apparent difference in the ability of stressed and unstressed cells to survive surface disinfection with the tested agents. Treatments to reduce viable E. coli O157:H7 cells on rocket leaves stored at 25°C were more effective than when used on those stored at 10°C. Washing with peroxyacetic acid or chlorine solution did not ensure the safety of rocket leaves, but such treatments could reduce the likelihood of water-mediated transfer of E. coli O157:H7 during washing and subsequent processing.

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