scholarly journals Insights into Genome Recoding from the Mechanism of a Classic +1-Frameshifting tRNA

2021 ◽  
Author(s):  
Howard Gamper ◽  
Haixing Li ◽  
Isao Masuda ◽  
D. Miklos Robkis ◽  
Thomas Christian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Conformational Change ◽  
Molecular Mechanism ◽  
Late Stage ◽  
Anticodon Loop ◽  
Pairing Mechanism ◽  
Extra Nucleotide ◽  
In Cells

ABSTRACTWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

scholarly journals Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Howard Gamper ◽  
Haixing Li ◽  
Isao Masuda ◽  
D. Miklos Robkis ◽  
Thomas Christian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Conformational Change ◽  
Molecular Mechanism ◽  
Late Stage ◽  
Anticodon Loop ◽  
Pairing Mechanism ◽  
Extra Nucleotide ◽  
In Cells

AbstractWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

scholarly journals Plasma albumin synthesis during neonatal development of the rat

1967 ◽  
Vol 102 (3) ◽  
pp. 760-762 ◽  
Author(s):  
R. W. Wise ◽  
I. T. Oliver
Keyword(s):  
Amino Acids ◽  
Specific Antibody ◽  
Late Stage ◽  
Normal Adult ◽  
Albumin Synthesis ◽  
Plasma Albumin ◽  
Pregnant Rat ◽  
Liver Slices ◽  
Parenchymal Cells ◽  
Very High

1. Liver slices were incubated with (14)C-labelled amino acids. Albumin was isolated from the slices by precipitation with specific antibody and the incorporated radioactivity measured. 2. The rate of synthesis was seen to be equal in liver slices from adult and late-stage foetal rats. 3. Synthesis was very high in the pregnant rat (three times the normal adult value) and in the 5-15-day post-natal rat (twice the normal adult value). 4. The post-natal increase may be due to the disappearance of haemopoietic tissue and its replacement by active parenchymal cells.

Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

1995 ◽  
Vol 14 (20) ◽  
pp. 4922-4931 ◽  
Author(s):  
G. Travé ◽  
P. J. Lacombe ◽  
M. Pfuhl ◽  
M. Saraste ◽  
A. Pastore
Keyword(s):  
Conformational Change ◽  
Molecular Mechanism ◽  
Ef Hands

Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids

1990 ◽  
Vol 97 (3) ◽  
pp. 479-485
Author(s):  
J.R. Jara ◽  
J.H. Martinez-Liarte ◽  
F. Solano ◽  
R. Penafiel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Initial Rate ◽  
Aromatic Amino Acids ◽  
Melanoma Cells ◽  
Transport Systems ◽  
Specific Amino Acid ◽  
Intracellular Content ◽  
Tyrosine Hydroxylation ◽  
In Cells

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks' isotonic medium or preloaded with a specific amino acid by 1 h preincubation in the same solution containing 2 mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the L1 system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.

scholarly journals Binding of L-branched-chain amino acids causes a conformational change in BkdR.

1997 ◽  
Vol 179 (1) ◽  
pp. 276-279 ◽  
Author(s):  
K T Madhusudhan ◽  
N Huang ◽  
E H Braswell ◽  
J R Sokatch
Keyword(s):  
Amino Acids ◽  
Conformational Change ◽  
Branched Chain Amino Acids ◽  
Branched Chain

scholarly journals ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

2019 ◽  
Vol 150 (5) ◽  
pp. 1022-1030 ◽  
Author(s):  
Dandan Xu ◽  
Weiwei Dai ◽  
Lydia Kutzler ◽  
Holly A Lacko ◽  
Leonard S Jefferson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Damage ◽  
Amino Acid ◽  
Protein Kinase ◽  
Dna Damage Response ◽  
Dependent Manner ◽  
Mouse Embryo Fibroblasts ◽  
Damage Response ◽  
Mtorc1 Activation ◽  
In Cells

ABSTRACT Background The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

scholarly journals A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)

2018 ◽  
Vol 29 (9) ◽  
pp. 1060-1074 ◽  
Author(s):  
Tomohiro Kubo ◽  
Yuqing Hou ◽  
Deborah A. Cochran ◽  
George B. Witman ◽  
Toshiyuki Oda
Keyword(s):  
Conformational Change ◽  
Molecular Mechanism ◽  
Wild Type ◽  
Sliding Direction ◽  
Large Conformational Change ◽  
Dynein Arms ◽  
Biochemical Analyses

Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.

scholarly journals Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Maren Kirstin Schuhmacher ◽  
Serap Beldar ◽  
Mina S. Khella ◽  
Alexander Bröhm ◽  
Jan Ludwig ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Histone H3 ◽  
Protein Methylation ◽  
Sequence Specificity ◽  
Sequence Design ◽  
Substrate Peptide ◽  
Lysine Methyltransferase ◽  
In Cells

AbstractSETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

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