scholarly journals A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 309-320 ◽  
Author(s):  
K Kawakami ◽  
S Pande ◽  
B Faiola ◽  
D P Moore ◽  
J D Boeke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Processing ◽  
Ribonuclease H ◽  
Wild Type ◽  
Ribosomal Frameshifting ◽  
Translational Frameshifting ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Ty1 Retrotransposition ◽  
Rate Limiting

Abstract Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.

scholarly journals The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

1992 ◽  
Vol 12 (7) ◽  
pp. 2986-2996 ◽  
Author(s):  
P Vreken ◽  
H A Raué
Keyword(s):  
Half Life ◽  
Rnase H ◽  
Start Codon ◽  
Wild Type ◽  
Coding Region ◽  
Endonucleolytic Cleavage ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Nuclease Mapping ◽  
Rate Limiting

Insertion of an 18-nucleotide-long poly(G) tract into the 3'-terminal untranslated region of yeast phosphoglycerate kinase (PGK1) mRNA increases its chemical half-life by about a factor of 2 (P. Vreken, R. Van der Veen, V. C. H. F. de Regt, A. L. de Maat, R. J. Planta, and H. A. Raué, Biochimie 73:729-737, 1991). In this report, we show that this insertion also causes the accumulation of a degradation intermediate extending from the poly(G) sequence down to the transcription termination site. Reverse transcription and S1 nuclease mapping experiments demonstrated that this intermediate is the product of shorter-lived primary fragments resulting from endonucleolytic cleavage immediately downstream from the U residue of either of two 5'-GGUG-3' sequences present between positions 1100 and 1200 close to the 3' terminus (position 1251) of the coding sequence. Similar endonucleolytic cleavages appear to initiate degradation of wild-type PGK1 mRNA. Insertion of a poly(G) tract just upstream from the AUG start codon resulted in the accumulation of a 5'-terminal degradation intermediate extending from the insertion to the 1100-1200 region. RNase H degradation in the presence of oligo(dT) demonstrated that the wild-type and mutant PGK1 mRNAs are deadenylated prior to endonucleolytic cleavage and that the half-life of the poly(A) tail is three- to sixfold lower than that of the remainder of the mRNA. Thus, the endonucleolytic cleavage constitutes the rate-limiting step in degradation of both wild-type and mutant PGK1 transcripts, and the resulting fragments are degraded by a 5'----3' exonuclease, which appears to be severely retarded by a poly(G) sequence.

scholarly journals DNA replication licensing in stem cells: Gatekeeping the commitment to proliferation

2018 ◽  
Vol 217 (5) ◽  
pp. 1563-1565
Author(s):  
Hilary A. Coller
Keyword(s):  
Stem Cells ◽  
Dna Replication ◽  
Intestinal Stem Cells ◽  
Wild Type ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cell Biol ◽  
Rate Limiting

Carroll et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201708023) developed a method to assess DNA replication licensing in tissues. They show that intestinal stem cells within wild-type crypts, but not in crypts with cancer-causing mutations, are largely unlicensed, suggesting that licensing may represent a rate-limiting step in the commitment to proliferation.

scholarly journals Is the 135S Poliovirus Particle an Intermediate during Cell Entry?

2000 ◽  
Vol 74 (18) ◽  
pp. 8757-8761 ◽  
Author(s):  
Yan Huang ◽  
James M. Hogle ◽  
Marie Chow
Keyword(s):  
Conformational Transition ◽  
Cell Entry ◽  
Wild Type ◽  
Cold Adapted ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
A Cell ◽  
Low Efficiency ◽  
Rate Limiting ◽  
Initial Conversion

ABSTRACT Poliovirus binding to its receptor (PVR) on the cell surface induces a conformational transition which generates an altered particle with a sedimentation value of 135S versus the 160S of the native virion. A number of lines of evidence suggest that the 135S particle is a cell entry intermediate. However, the low infection efficiencies of the 135S particle and the absence of detectable 135S particles during infection at 26°C by the cold-adapted mutants argue against a role for the 135S particle during the cell entry process. We show here that binding of 135S-antibody complexes to the Fc receptor (CDw32) increases the infectivity of these particles by 2 to 3 orders of magnitude. Thus, the low efficiency of infection by 135S particles is due in part to the low binding affinity of these particles. In addition, we show that there is an additional stage in the entry process that is associated with RNA release. This stage occurs after formation of the 135S particle, is rate limiting during infection at 37°C, but not at 26°C, and is PVR independent. The data also demonstrate that during infection at 26°C, the rate-limiting step is the PVR-mediated conversion of wild-type 160S particles to 135S particles. This suggests that during infection at 26°C by the cold-adapted viruses, 135S particles are formed, but they fail to accumulate to detectable levels because the subsequent post-135S particle events occur at a significantly faster rate than the initial conversion of 160S to 135S particles. These data support a model in which the 135S particle is an intermediate during poliovirus entry.

Methionine and Alanine Substitutions Show That the Formation of Wild-Type-like Structure in the Carboxy-Terminal Domain of T4 Lysozyme Is a Rate-Limiting Step in Folding†

Biochemistry ◽  
1999 ◽  
Vol 38 (44) ◽  
pp. 14451-14460 ◽  
Author(s):  
Nadine C. Gassner ◽  
Walter A. Baase ◽  
Joel D. Lindstrom ◽  
Jirong Lu ◽  
Frederick W. Dahlquist ◽  
...  
Keyword(s):  
T4 Lysozyme ◽  
Wild Type ◽  
Terminal Domain ◽  
Rate Limiting Step ◽  
Carboxy Terminal Domain ◽  
Limiting Step ◽  
Carboxy Terminal ◽  
Rate Limiting

scholarly journals Mutagenesis and mechanistic study of a glycoside hydrolase family 54 α-L-arabinofuranosidase from Trichoderma koningii

2006 ◽  
Vol 401 (2) ◽  
pp. 551-558 ◽  
Author(s):  
Chin-Feng Wan ◽  
Wei-Hong Chen ◽  
Cheng-Ta Chen ◽  
Margaret Dah-Tsyr Chang ◽  
Lee-Chiang Lo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Enzymatic Reaction ◽  
Glycoside Hydrolase Family ◽  
Wild Type ◽  
Trichoderma Koningii ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hydrolase Family ◽  
Enzyme Intermediate

A GH (glycoside hydrolase) family 54 α-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii α-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl–enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl–enzyme intermediate was proposed. Based on the kcat values of a series of aryl-α-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, βlg=−0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pKa values (>6) of leaving phenols, with βlg=−1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

scholarly journals Basis for discrimination by engineered CRISPR/Cas9 enzymes

2019 ◽  
Author(s):  
Mu-Sen Liu ◽  
Shanzhong Gong ◽  
Helen-Hong Yu ◽  
Kyungseok Jung ◽  
Kenneth A. Johnson ◽  
...  
Keyword(s):  
High Fidelity ◽  
Dna Unwinding ◽  
Biological Applications ◽  
Wild Type ◽  
Guide Rna ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Target Dna ◽  
Rate Limiting

AbstractCRISPR/Cas9 is a programmable genome editing tool that has been widely used for biological applications. While engineered Cas9s have been reported to increase discrimination against off-target cleavage compared with wild type Streptococcus pyogenes (SpCas9) in vivo, the mechanism for enhanced specificity has not been extensively characterized. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. While DNA unwinding is the rate-limiting step for on-target cleavage by SpCas9, we show that chemistry is seriously impaired by more than 100-fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the rate of chemistry without increasing the rate of DNA rewinding—the kinetic partitioning favors release rather than cleavage of a bound off-target substrate because chemistry is slow. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region

1992 ◽  
Vol 12 (7) ◽  
pp. 2986-2996
Author(s):  
P Vreken ◽  
H A Raué
Keyword(s):  
Half Life ◽  
Rnase H ◽  
Start Codon ◽  
Wild Type ◽  
Coding Region ◽  
Endonucleolytic Cleavage ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Nuclease Mapping ◽  
Rate Limiting

Insertion of an 18-nucleotide-long poly(G) tract into the 3'-terminal untranslated region of yeast phosphoglycerate kinase (PGK1) mRNA increases its chemical half-life by about a factor of 2 (P. Vreken, R. Van der Veen, V. C. H. F. de Regt, A. L. de Maat, R. J. Planta, and H. A. Raué, Biochimie 73:729-737, 1991). In this report, we show that this insertion also causes the accumulation of a degradation intermediate extending from the poly(G) sequence down to the transcription termination site. Reverse transcription and S1 nuclease mapping experiments demonstrated that this intermediate is the product of shorter-lived primary fragments resulting from endonucleolytic cleavage immediately downstream from the U residue of either of two 5'-GGUG-3' sequences present between positions 1100 and 1200 close to the 3' terminus (position 1251) of the coding sequence. Similar endonucleolytic cleavages appear to initiate degradation of wild-type PGK1 mRNA. Insertion of a poly(G) tract just upstream from the AUG start codon resulted in the accumulation of a 5'-terminal degradation intermediate extending from the insertion to the 1100-1200 region. RNase H degradation in the presence of oligo(dT) demonstrated that the wild-type and mutant PGK1 mRNAs are deadenylated prior to endonucleolytic cleavage and that the half-life of the poly(A) tail is three- to sixfold lower than that of the remainder of the mRNA. Thus, the endonucleolytic cleavage constitutes the rate-limiting step in degradation of both wild-type and mutant PGK1 transcripts, and the resulting fragments are degraded by a 5'----3' exonuclease, which appears to be severely retarded by a poly(G) sequence.

scholarly journals Identification of a Human Decapping Complex Associated with hUpf Proteins in Nonsense-Mediated Decay

2002 ◽  
Vol 22 (23) ◽  
pp. 8114-8121 ◽  
Author(s):  
Jens Lykke-Andersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Premature Termination ◽  
The Other ◽  
Nonsense Mediated Decay ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Premature Termination Codons ◽  
Rate Limiting ◽  
Decapping Enzymes

ABSTRACT Decapping is a key step in general and regulated mRNA decay. In Saccharomyces cerevisiae it constitutes a rate-limiting step in the nonsense-mediated decay pathway that rids cells of mRNAs containing premature termination codons. Here two human decapping enzymes are identified, hDcp1a and hDcp2, as well as a homolog of hDcp1a, termed hDcp1b. Transiently expressed hDcp1a and hDcp2 proteins localize primarily to the cytoplasm and form a complex in human cell extracts. hDcp1a and hDcp2 copurify with decapping activity, an activity sensitive to mutation of critical hDcp residues. Importantly, coimmunoprecipitation assays demonstrate that hDcp1a and hDcp2 interact with the nonsense-mediated decay factor hUpf1, both in the presence and in the absence of the other hUpf proteins, hUpf2, hUpf3a, and hUpf3b. These data suggest that a human decapping complex may be recruited to mRNAs containing premature termination codons by the hUpf proteins.

scholarly journals Insight into wild-type and T1372E TET2-mediated 5hmC oxidation using ab initio QM/MM calculations

2018 ◽  
Vol 9 (44) ◽  
pp. 8433-8445 ◽  
Author(s):  
Hedieh Torabifard ◽  
G. Andrés Cisneros
Keyword(s):  
Ab Initio ◽  
Wild Type ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Insight Into

T1372E TET2 stalls at 5hmC due to unfavorable orientation of substrate, which increases barrier of the rate limiting step.

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