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.

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 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 Massive Factorial Design Untangles Coding Sequences Determinants of Translation Efficacy

2017 ◽  
Author(s):  
Guillaume Cambray ◽  
Joao C. Guimaraes ◽  
Adam Paul Arkin
Keyword(s):  
Large Scale ◽  
Small Sample ◽  
Translation Efficiency ◽  
E Coli ◽  
Synthetic Dna ◽  
Critical Design ◽  
Codon Composition ◽  
Expression Activity ◽  
Small Sample Sizes ◽  
Epigenetic Disruption

AbstractComparative analyses of natural sequences or variant libraries are often used to infer mechanisms of expression, activity and evolution. Contingent selective histories and small sample sizes can profoundly bias such approaches. Both limitations can be lifted using precise design of large-scale DNA synthesis. Here, we precisely design 5E. coligenomes worth of synthetic DNA to untangle the relative contributions of 8 interlaced sequence properties described independently as major determinants of translation inEscherichia coli. To expose hierarchical effects, we engineer an inducible translational coupling device enabling epigenetic disruption of mRNA secondary structures. We find that properties commonly believed to modulate translation generally explain less than a third of the variation in protein production. We describe dominant effects of mRNA structures over codon composition on both initiation and elongation, and previously uncharacterized relationships among factors controlling translation. These results advance our understanding of translation efficiency and expose critical design challenges.

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.

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

2020 ◽  
Author(s):  
Chaoyong Huang ◽  
Liwei Guo ◽  
Jingge Wang ◽  
Ning Wang ◽  
Yi-Xin Huo
Keyword(s):  
Metabolic Engineering ◽  
Large Scale ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Open Reading Frames ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
E Coli ◽  
Stable Plasmid ◽  
Reading Frames

ABSTRACTBacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

scholarly journals Molecular Characterization and Postsplicing Fate of Three Introns within the Single rRNA Operon of the Hyperthermophilic ArchaeonAeropyrum pernix K1

1998 ◽  
Vol 180 (14) ◽  
pp. 3635-3643 ◽  
Author(s):  
Norimichi Nomura ◽  
Yoshihiko Sako ◽  
Aritsune Uchida
Keyword(s):  
Sequence Data ◽  
Protein Translation ◽  
Open Reading Frames ◽  
Rrna Operon ◽  
Rrna Genes ◽  
23S Rrna ◽  
Homing Endonucleases ◽  
Two Dimensional Gel Electrophoresis ◽  
E Coli

ABSTRACT The single rRNA operon (arnS-arnL) of the hyperthermophilic archaeon Aeropyrum pernix K1 was sequenced. The DNA sequence data and detailed RNA analyses disclosed an unusual feature: the presence of three introns at hitherto undescribed insertion positions within the rRNA genes. The 699-nucleotide (nt) intron Iα was located at position 908 (Escherichia coli numbering [H. F. Noller, Annu. Rev. Biochem. 53:119–162, 1984]) of the 16S rRNA, while the 202-nt intron Iβ and 575-nt intron Iγ were located at positions 1085 and 1927 (E. coli numbering), respectively, of the 23S rRNA. They were located within highly conserved sites which have been implicated as crucial for rRNA function in E. coli. All three introns were remarkably AT rich (41.5 to 43.1 mol% G+C) compared with the mature rRNAs (67.7 and 69.2 mol% G+C for 16S and 23S rRNAs, respectively). No obvious primary sequence similarities were detected among them. After splicing from rRNA transcripts in vivo, a large quantity of intronic RNAs were stably retained in the linear monomeric form, whereas a trace of topoisomeric RNA molecules also appeared, as characterized by their behavior in two-dimensional gel electrophoresis. Secondary structural models of the Iα-, Iβ-, and Iγ-containing rRNA precursors agree with the bulge-helix-bulge motif. Two of the introns, Iα and Iγ, contained open reading frames whose protein translation exhibited no overall similarity with proteins reported so far. However, both share a LAGLI-DADG motif characteristic of homing endonucleases.

scholarly journals Molecular evolution of the Escherichia coli chromosome. I. Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 345-358
Author(s):  
A Stoltzfus ◽  
J F Leslie ◽  
R Milkman
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Natural Variation ◽  
Genetic Recombination ◽  
Coli Strain ◽  
Open Reading Frames ◽  
Dna Rearrangements ◽  
E Coli ◽  
Wild Strains ◽  
Reading Frames

Abstract We present the sequence of a 3500-bp region of the Escherichia coli strain K12 chromosome lying between the tryptophan operon and the tonB gene. Analysis of the sequence yields six open reading frames that have properties characteristic of genes for proteins. The reading frames are closely spaced, and putative transcription units and control sites compose over 95% of the DNA. The sequences of several wild strains of E. coli have been determined for a large segment of the region described. Comparison of these sequences reveals the effects of base substitutions, DNA rearrangements, and recombination. In the regions presumably expressed as polypeptides, most of the natural variation results from synonymous substitutions. However, the DNA rearrangements identified have end points within the open reading frames and disrupt them in a variety of ways. The effects of genetic recombination between strains, recently found to be significant on a large scale in E. coli, are also apparent in the region between trp and tonB.

scholarly journals Scikit-ribo: Accurate estimation and robust modeling of translation dynamics at codon resolution

2017 ◽  
Author(s):  
Han Fang ◽  
Yi-Fei Huang ◽  
Aditya Radhakrishnan ◽  
Adam Siepel ◽  
Gholson J. Lyon ◽  
...  
Keyword(s):  
Open Source Software ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Accurate Estimation ◽  
Translation Efficiency ◽  
Sampling Errors ◽  
Genome Wide ◽  
Rnaseq Data ◽  
A Site ◽  
Level Analysis

AbstractRibosome profiling (Riboseq) is a powerful technique for measuring protein translation, however, sampling errors and biological biases are prevalent and poorly understand. Addressing these issues, we present Scikit-ribo (https://github.com/hanfang/scikit-ribo), the first open-source software for accurate genome-wide A-site prediction and translation efficiency (TE) estimation from Riboseq and RNAseq data. Scikit-ribo accurately identifies A-site locations and reproduces codon elongation rates using several digestion protocols (r = 0.99). Next we show commonly used RPKM-derived TE estimation is prone to biases, especially for low-abundance genes. Scikit-ribo introduces a codon-level generalized linear model with ridge penalty that correctly estimates TE while accommodating variable codon elongation rates and mRNA secondary structure. This corrects the TE errors for over 2000 genes in S. cerevisiae, which we validate using mass spectrometry of protein abundances (r = 0.81) and allows us to determine the Kozak-like sequence directly from Riboseq. We conclude with an analysis of coverage requirements needed for robust codon-level analysis, and quantify the artifacts that can occur from cycloheximide treatment.

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.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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