scholarly journals Kinetic study in vitro of Escherichia coli promoter closure during transcription initiation

1995 ◽  
Vol 306 (1) ◽  
pp. 123-128 ◽  
Author(s):  
B Schmitt ◽  
C Reiss
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Beta Lactamase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Run Off ◽  
Lactamase Gene ◽  
Rate Limiting ◽  
Coli Promoter

The rate of closure of two Escherichia coli promoters borne by plasmid pBR322, following transcription initiation from the open complex, was probed in vitro by the protection of unpaired thymines in the open complex against oxidation by KMnO4. Run-off transcription kinetics were also studied under identical conditions. Closure of the open promoter appears to be by far the rate-limiting step of transcription initiation and elongation for the linearized beta-lactamase gene, and is strongly dependent on template topology for the RNAI gene. It is suggested that the corresponding signals are deposited 30 bases at least downstream of transcription initiation and that promoter closure, and its clearance by elongating RNA polymerase, may occur almost simultaneously.

scholarly journals Escherichia coli Peptidase A, B, or N Can Process Translation Inhibitor Microcin C

2008 ◽  
Vol 190 (7) ◽  
pp. 2607-2610 ◽  
Author(s):  
Teymur Kazakov ◽  
Gaston H. Vondenhoff ◽  
Kirill A. Datsenko ◽  
Maria Novikova ◽  
Anastasia Metlitskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Trna Synthetase ◽  
Methionine Residue ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Translation Inhibitor ◽  
Rate Limiting ◽  
Broad Specificity

ABSTRACT The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

scholarly journals Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 153 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Rubisco Activase ◽  
Power Transfer ◽  
Target Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Priya Bariya ◽  
Linda L. Randall
Keyword(s):  
Lipid Bilayers ◽  
Rate Constant ◽  
Apparent Rate Constant ◽  
Membrane Vesicles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secretory System ◽  
Rate Limiting

ABSTRACTIn all cells, a highly conserved channel transports proteins across membranes. InEscherichia coli, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing threein vitrosystems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations madein vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCEThis work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the threein vitrosystems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.

scholarly journals DETERMINATION OF l-HISTIDINE IN NANOGRAM AMOUNTS AND THE QUESTION OF ITS OCCURRENCE IN MAST CELLS

1972 ◽  
Vol 20 (11) ◽  
pp. 917-922 ◽  
Author(s):  
DAVID I. WILKINSON ◽  
DAVID GLICK
Keyword(s):  
Mast Cells ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Peritoneal Mast Cells ◽  
Before And After ◽  
Chromatographic Detection ◽  
Rat Peritoneal Mast Cells ◽  
Rate Limiting

In an attempt to clarify the question of whether histidine is stored in the mast cell for coversion to histamine or whether the rate of conversion is rapid enough to prevent accumulation of histidine so that the rate-limiting step is the histidine uptake, it was found that no histidine was demonstrable in rat peritoneal mast cells by either quantitative analysis or paper chromatographic detection. Microadaptation of Hassall's method, based on conversion of l-histidine by histidase to urocanic acid and measurement of the latter by its absorbance at 277 nm, was made to permit determination of histidine in nanogram amounts in the presence of histamine. This adaptation was found reliable when compared with the o-phthalaldehyde method in estimation of l-histidine in serum and in insulin hydrolysate, and then it was applied to analysis of mast cells before and after l-histidine uptake in vitro. The adaptation should be generally useful in microanalysis of l-histidine in histologically and cytologically defined samples.

scholarly journals Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose

1976 ◽  
Vol 156 (2) ◽  
pp. 477-480 ◽  
Author(s):  
D Herbert ◽  
H L Kornberg
Keyword(s):  
Escherichia Coli ◽  
Continuous Culture ◽  
Glucose Transport ◽  
Growth Rates ◽  
Glucose Limitation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Wide Range ◽  
Rate Limiting

Over a wide range of growth rates, two strains of Escherichia coli growing aerobically in continuous culture under glucose limitation utilized glucose at rates identical with those at which cells harvested from the chemostats transported [14C]glucose.

scholarly journals The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors

1982 ◽  
Vol 203 (2) ◽  
pp. 505-510 ◽  
Author(s):  
R H Jackson ◽  
J A Cole ◽  
A Cornish-Bowden
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Maximum Velocity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Cytochrome C Reduction ◽  
Pattern Of Variation ◽  
Rate Limiting

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.

scholarly journals Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered Escherichia coli without Loss of Specific Isotopic Enrichment

2003 ◽  
Vol 69 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Maria-Manuel Sampaio ◽  
Helena Santos ◽  
Winfried Boos
Keyword(s):  
Escherichia Coli ◽  
Cold Water ◽  
Genetically Engineered ◽  
Isotopic Enrichment ◽  
Rhodothermus Marinus ◽  
Phosphotransferase System ◽  
Gene Encoding ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

ABSTRACT We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-α-d-mannosyl-d-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-α-d-mannosyl-d-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-α-d-mannosyl-d-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-α-d-mannosyl-d-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.

scholarly journals Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

1990 ◽  
Vol 265 (3) ◽  
pp. 899-902 ◽  
Author(s):  
T R Hawkes ◽  
T Lewis ◽  
J R Coggins ◽  
D M Mousdale ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Lag Phase ◽  
Chorismate Synthase ◽  
Phosphate Release ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state ‘burst’ or ‘lag’ phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

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