scholarly journals The -11A of promoter DNA and two conserved amino acids in the melting region of  70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact

2007 ◽  
Vol 35 (12) ◽  
pp. 4141-4153 ◽  
Author(s):  
L. A. Schroeder ◽  
A.-J. Choi ◽  
P. L. deHaseth
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Melting Region ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Rate Limiting ◽  
Rna Polymerase Promoter ◽  
Conserved Amino Acids

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

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 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.

Substrate-assisted mechanism of catalytic hydrolysis of misaminoacylated tRNA required for protein synthesis fidelity

2019 ◽  
Vol 476 (4) ◽  
pp. 719-732 ◽  
Author(s):  
Mykola M. Ilchenko ◽  
Mariia Yu. Rybak ◽  
Alex V. Rayevsky ◽  
Oksana P. Kovalenko ◽  
Igor Ya. Dubey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Mechanism ◽  
Thermus Thermophilus ◽  
Extensive Study ◽  
Water Molecules ◽  
Amino Acid Residues ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Mechanism Of Hydrolysis ◽  
Rate Limiting

Abstract d-aminoacyl-tRNA-deacylase (DTD) prevents the incorporation of d-amino acids into proteins during translation by hydrolyzing the ester bond between mistakenly attached amino acids and tRNAs. Despite extensive study of this proofreading enzyme, the precise catalytic mechanism remains unknown. Here, a combination of biochemical and computational investigations has enabled the discovery of a new substrate-assisted mechanism of d-Tyr-tRNATyr hydrolysis by Thermus thermophilus DTD. Several functional elements of the substrate, misacylated tRNA, participate in the catalysis. During the hydrolytic reaction, the 2′-OH group of the А76 residue of d-Tyr-tRNATyr forms a hydrogen bond with a carbonyl group of the tyrosine residue, stabilizing the transition-state intermediate. Two water molecules participate in this reaction, attacking and assisting ones, resulting in a significant decrease in the activation energy of the rate-limiting step. The amino group of the d-Tyr aminoacyl moiety is unprotonated and serves as a general base, abstracting the proton from the assisting water molecule and forming a more nucleophilic ester-attacking species. Quantum chemical methodology was used to investigate the mechanism of hydrolysis. The DFT-calculated deacylation reaction is in full agreement with the experimental data. The Gibbs activation energies for the first and second steps were 10.52 and 1.05 kcal/mol, respectively, highlighting that the first step of the hydrolysis process is the rate-limiting step. Several amino acid residues of the enzyme participate in the coordination of the substrate and water molecules. Thus, the present work provides new insights into the proofreading details of misacylated tRNAs and can be extended to other systems important for translation fidelity.

RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene

1991 ◽  
Vol 11 (10) ◽  
pp. 5285-5290
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Intermediate Level ◽  
Hsp70 Gene ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Initiation Of Transcription ◽  
Rate Limiting

An RNA polymerase II molecule is associated with the 5' end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides (A. E. Rougvie and J. T. Lis, Cell 54:795-804, 1988). Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here we report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5' end of hsp70 in cells exposed to the intermediate heat shock temperatures of 27 and 30 degrees C. At 30 degrees C, each copy of hsp70 was transcribed approximately five times during the 25-min heat shock that we used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

scholarly journals Recruitment of RNA Polymerase Is a Rate-limiting Step for the Activation of the ς54PromoterPuofPseudomonas putida

1999 ◽  
Vol 274 (47) ◽  
pp. 33790-33794 ◽  
Author(s):  
Manuel Carmona ◽  
Vı́ctor de Lorenzo ◽  
Giovanni Bertoni
Keyword(s):  
Rna Polymerase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

scholarly journals Simultaneous quantification of depolymerization and mineralization rates by a novel <sup>15</sup>N tracing model

SOIL ◽  
2016 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Louise C. Andresen ◽  
Anna-Karin Björsne ◽  
Samuel Bodé ◽  
Leif Klemedtsson ◽  
Pascal Boeckx ◽  
...  
Keyword(s):  
Amino Acids ◽  
Terrestrial Ecosystems ◽  
Organic N ◽  
N Availability ◽  
Simultaneous Quantification ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Use Efficiency ◽  
Soil N Cycle ◽  
Rate Limiting

Abstract. The depolymerization of soil organic matter, such as proteins and (oligo-)peptides, into monomers (e.g. amino acids) is currently considered to be the rate-limiting step for nitrogen (N) availability in terrestrial ecosystems. The mineralization of free amino acids (FAAs), liberated by the depolymerization of peptides, is an important fraction of the total mineralization of organic N. Hence, the accurate assessment of peptide depolymerization and FAA mineralization rates is important in order to gain a better process-based understanding of the soil N cycle. In this paper, we present an extended numerical 15N tracing model Ntrace, which incorporates the FAA pool and related N processes in order to provide a more robust and simultaneous quantification of depolymerization and gross mineralization rates of FAAs and soil organic N. We discuss analytical and numerical approaches for two forest soils, suggest improvements of the experimental work for future studies, and conclude that (i) when about half of all depolymerized peptide N is directly mineralized, FAA mineralization can be as important a rate-limiting step for total gross N mineralization as peptide depolymerization rate; (ii) gross FAA mineralization and FAA immobilization rates can be used to develop FAA use efficiency (NUEFAA), which can reveal microbial N or carbon (C) limitation.

scholarly journals Depolymerization and mineralization – investigating N availability by a novel <sup>15</sup>N tracing model

2016 ◽  
Author(s):  
Louise C. Andresen ◽  
Anna-Karin Björsne ◽  
Samuel Bodé ◽  
Leif Klemedtsson ◽  
Pascal Boeckx ◽  
...  
Keyword(s):  
Amino Acids ◽  
N Mineralization ◽  
Soil Disturbance ◽  
Analytical Models ◽  
N Availability ◽  
Total N ◽  
15N Pool Dilution ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract. Depolymerization of soil organic matter such as proteins and peptides into monomers (e.g. amino acids) is currently thought to be the rate limiting step for N availability in terrestrial N cycles. The mineralization of free amino acids (FAA), liberated by depolymerization of peptides, is an important fraction of the total N mineralization. Accurate assessment 10 of peptide depolymerization and FAA mineralization rates is important in order to gain a better understanding of the N cycle dynamics. Due to the short time span, soil disturbance and unnatural high FAA content during the first few hours after the labelling with the traditional 15N pool dilution experiments, analytical models might overestimate peptide depolymerization rate. In this paper, we present an extended numerical 15N tracing model Ntrace which incorporates the FAA pool and related N processes in order to 1) provide a more robust and coherent estimation of production and mineralization rates of FAAs; 2) 15 and 2) suggest an amino acid N use efficiency (NUEFAA) for soil microbes, which is a more realistic estimation of soil microbial NUE compared to the NUE estimated by analytical methods. We compare analytical and numerical approaches for two forest soils; suggest improvements of the experimental work for future studies; and conclude that: i) FAA mineralization might be as equally an important rate limiting step for gross N mineralization as peptide depolymerization rate is, because about half of all depolymerized peptide N is consecutively being mineralized; and that ii) FAA mineralization and FAA 20 immobilization rates should be used for assessing NUEFAA.

scholarly journals DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37°C and results in the accumulation of an off-pathway intermediate

2009 ◽  
Vol 37 (16) ◽  
pp. 5390-5404 ◽  
Author(s):  
Anastasia Rogozina ◽  
Evgeny Zaychikov ◽  
Malcolm Buckle ◽  
Hermann Heumann ◽  
Bianca Sclavi
Keyword(s):  
Rna Polymerase ◽  
Dna Melting ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting
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