scholarly journals Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in Bacillus subtilis

2020 ◽  
Author(s):  
Laura E. Ritchey ◽  
David C. Tack ◽  
Helen Yakhnin ◽  
Elizabeth A. Jolley ◽  
Sarah M. Assmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Protein Binding ◽  
Rna Structure ◽  
Transcription Termination ◽  
Dimethyl Sulfate ◽  
Transcript Abundance ◽  
Amino Acid Starvation ◽  
Leader Peptide

ABSTRACTRNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.

scholarly journals Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in Bacillus subtilis

RNA ◽  
2020 ◽  
Vol 26 (10) ◽  
pp. 1431-1447
Author(s):  
Laura E. Ritchey ◽  
David C. Tack ◽  
Helen Yakhnin ◽  
Elizabeth A. Jolley ◽  
Sarah M. Assmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Structure ◽  
Amino Acid Starvation ◽  
Rna Switches

Autophagy is essential for the maintenance of amino acids and ATP levels during acute amino acid starvation in MDAMB231 cells

2018 ◽  
Vol 36 (2) ◽  
pp. 65-79 ◽  
Author(s):  
Mark Thomas ◽  
Tanja Davis ◽  
Ben Loos ◽  
Balindiwe Sishi ◽  
Barbara Huisamen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Starvation ◽  
Atp Levels

scholarly journals The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels

2020 ◽  
Vol 48 (6) ◽  
pp. 3071-3088
Author(s):  
Matthew R McFarland ◽  
Corina D Keller ◽  
Brandon M Childers ◽  
Stephen A Adeniyi ◽  
Holly Corrigall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurological Disorders ◽  
Trna Synthetase ◽  
Northern Blotting ◽  
Amino Acid Starvation ◽  
Starvation Response ◽  
Kinase Activation ◽  
Trna Synthetases ◽  
Starvation Conditions

Abstract During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

scholarly journals Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown

1976 ◽  
Vol 154 (2) ◽  
pp. 541-552
Author(s):  
J E. M. Midgley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Turnover Rate ◽  
Amino Acid Starvation ◽  
High Rate ◽  
Glucose Medium ◽  
Amino Acid Deprivation ◽  
Acid Accumulation ◽  
Associated Proteins ◽  
Kinetics Of

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing “stepdown” in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active β-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in “stepped-down” rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of β-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.

scholarly journals Sequence of the N-terminal half of Bacillus amyloliquefaciens α-amylase

1980 ◽  
Vol 185 (2) ◽  
pp. 387-395 ◽  
Author(s):  
H Chung ◽  
F Friedberg
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Primary Structure ◽  
Bacillus Amyloliquefaciens ◽  
Proteolytic Enzymes ◽  
Alpha Amylase ◽  
Sequence Determination ◽  
N Terminus

Bacillus amyloliquefaciens alpha-amylase (1,4-alpha-D-glucan glucanohydrolase. EC 3.2.1.1), which is commercially supplied as ‘Bacillus subtilis alpha-amylase’ does not cross-react immunologically with B. subtilis alpha-amylase. This enzyme (from B. amyloliquefaciens) was cleaved by treatment with CNBr into seven fragments. Peptide A was selected for sequence determination. It is the longest one, containing 185 amino acids (i.e. approx. 50% of the total molecule) and connects to the hexapeptide of the N-terminus. Its primary structure was aligned by use of various proteolytic enzymes. The sequence of amino acids 181-184 is identical with that of amino acids 14-17 of the alpha-amylase isolated from B. subtilis (except that amino acid 183 is asparagine rather than aspartic acid).

scholarly journals An antibiotic-sensing leader peptide regulates translation and premature Rho-dependent transcription termination of thetopAIgene inEscherichia coli

2019 ◽  
Author(s):  
Gabriele Baniulyte ◽  
Joseph T. Wade
Keyword(s):  
Conformational Changes ◽  
Rna Structure ◽  
Transcription Termination ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Translational Repression ◽  
Leader Peptide ◽  
Rna Structures ◽  
Ribosome Stalling ◽  
Specific Manner

AbstractLong 5′ UTRs in bacteria often contain regulatory elements that modulate expression of the downstream gene in response to environmental stimuli. In most examples of such regulation, the mechanism involves switching between alternative 5′ UTR RNA structures that impact transcription, stability, or translation of the mRNA. Here, we show that transcription of theEscherichia coli topAIgene is prematurely terminated by the termination factor Rho under standard laboratory growth conditions, and that this occurs as a result of translational repression. Regulation oftopAItranslation is controlled by a sensory ORF,toiL, located within thetopAI5′ UTR. We show that ribosomes translatingtoiLstall in a sequence-specific manner in the presence of specific ribosome-targeting antibiotics. Ribosome stalling attoiLinduces conformational changes in the RNA structure of thetopAI5′ UTR, unmasking thetopAIribosome-binding site, thereby relieving translational repression and preventing premature transcription termination. Thus,toiLacts as a sensor of translation stress, leading to regulation oftopAIat both the translational and transcriptional levels.

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

1987 ◽  
Author(s):  
Richard J Jenny ◽  
Debra D Pittman ◽  
John J Toole ◽  
Ronald W Kriz ◽  
Randal J Kaufman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Human Factor ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Factor V ◽  
Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

Control of RNA Synthesis by Amino Acids in Landschutz Ascites Tumor Cells

1974 ◽  
Vol 52 (10) ◽  
pp. 867-876 ◽  
Author(s):  
Paul Jolicoeur ◽  
Fernand Labrie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Amino Acid Starvation ◽  
Cytoplasmic Rna ◽  
Polysomal Mrna ◽  
Deficient Medium ◽  
Synthesis And Processing

Landschutz cells incubated in amino acid-deficient medium for 2.5 h show a markedly reduced incorporation of [3H]uridine into 18 S and 28 S cytoplasmic ribosomal RNA (rRNA) and into 28 S, 32 S, and 36 S nuclear RNA measured during the last 90 min of incubation, whereas the radioactivity associated with 45 S pre-rRNA is not affected. Ten-minute pulse-labeling and 15-min pulse-chase experiments show that amino acid starvation inhibits both the synthesis and processing of 45 S pre-rRNA. Amino acid starvation has no significant effect on the labeling of the nucleotide pools. This effect of amino acids was specific for rRNA since the synthesis of 4 S and 5 S cytoplasmic RNA separated on polyacrylamide gels and of polysomal mRNA analyzed on sucrose gradients was not significantly affected during amino acid starvation. These data also indicate that RNA synthesis is non-coordinated in Landschutz cells. Among the 13 amino acids essential for growth of these cells, arginine and glutamine appear to be mainly responsible for the inhibition of synthesis of 18 S and 28 S rRNA measured during incubation in complete amino acid-deficient medium. The removal of any one of the other amino acids has a small inhibitory effect on the incorporation of [3H]uridine into rRNA and their effect on the synthesis of 18 S rRNA is more pronounced than on that of 28 S rRNA. Such effect results in an unbalanced production of these two ribosomal RNA species.

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