[9] Conformational analysis of tRNA

Ishida, T.; Arceneaux, J. Lincoln; Sueoka, Noboru

doi:10.1016/s0076-6879(71)20011-2

Cited by 10 publications

(10 citation statements)

References 8 publications

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“…Unfractionated aminoacyl-tRNA synthetases were prepared from E. coli MRE 600 and subjected to DEAE-cellulose chromatography before use (10). Normal and altered tRNAtrp species were routinely renatured before charging with tryptophan by heating at 50°C for 10 min in 0.1 M tris-HCl buffer (pH 7.8), 0.01 M Mg acetate ( 11,12). Uncharged labelled tRNAphe was recovered from the salt wash fraction obtained during the isolation of trp-tRNAtrP on BD-cellulose.…”

Section: Azir Valrmentioning

confidence: 99%

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

Buck

Griffiths

1982

Nucl Acids Res

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E. coli growing in the presence of iron-binding proteins produced tRNAtrp and tRNAphe molecules containing i6A instead of ms2i6A adjacent to the anticodon. These undermodified tRNAs functioned less efficiently than the fully modified molecules when translating synthetic polynucleotides containing contiguous codons in an in vitro system, but did not limit the translation of MS2 RNA. We examined the possibility that the altered tRNAs with lowered translational efficiencies could relieve transcription termination at the trp and phe attenuators and lead to increased operon expression under iron restricted conditions. Using trpR mutants we found that there was indeed greater expression of the trp operon during iron restricted growth. This increase was attributable solely to the tRNA alteration induced by iron restriction.

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Section: Azir Valrmentioning

confidence: 99%

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

Buck

Griffiths

1982

Nucl Acids Res

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“…But the graph is not rectilinear and can be approximated by the sum of two straight lines; for this case It means that our Phe-tRNAPhe preparation contains two kinds of molecules in the ratio y1Iy2. One of them We supposed that the existence of two different conformers of tRNA [14] is the source of this heterogeneity. As the equilibrium of tRNA conformers is known to depend on Mg2+ ions, we subjected our Phe-tRNAPh' samples to heating in solution at 48 "C for 3 min with 1 mM Mg2+ or alternatively with 5 mM EDTA to sequester all magnesium ions.…”

Section: Measurements Of the Affinity Constanl Of Phe-trnaph' To The mentioning

confidence: 99%

The Mechanism of Codon-Anticodon Interaction in Ribosomes. Heterogeneity of tRNA Complexes with 70-S Ribosomes of Escherichia coli

et al. 1978

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It was shown that at 20 mM Mg2+ and 0 or 20 "C each 70-S ribosome binds non-enzymatically about one Phe-tRNAPhe molecule in the A site if poly(U) is used as messenger. Only a minor amount of ribosomes (never exceeding 15 %) and after long incubation binds a second molecule of Phe-tRNAPhe and forms diphenylalanyl-tRNA. If the Phe-tRNAPhe preparation contains nonspecific deacylated tRNA, the latter is always bound in the P site up to one molecule per 70-S ribosome.The association constant for this non-specific tRNA binding is equal to (1.4 k 0.3) x lo7 M-' and is independent of the messenger poly(U) and the fiilling of the A site by a specific aminoacyl-tRNA.It was found that the codon-dependent binding of Phe-tRNAPhe in the A site is reversible, but two different types of complexes are formed with drastic differences of binding constants. This heterogeneity is due to the presence of two conformers of Phe-tRNAPhe. The equilibrium of the two Phe-tRNAPhe conformers depends on Mg2+ and can be shifted if tRNA is heated and Mg2' added or sequestered by complexing agents. One type of Phe-tRNAPhe yields complexes with 70-S ribosomes showing extremely high affinity constants (higher than 10" M -I ) . The second conformer is characterized by a comparatively low binding constant about 5 x lo6 M-I. In the binding of both species to 3 0 3 subunits no difference is seen.A big difference in the behaviour of both Phe-tRNAPhe conformers is found also in the absence of messenger RNA. The association constant of Phe-tRNAPhe (high-affinity conformer) to 7 0 3 ribosomes is about 3 x 10' M-' , but for the low-affinity conformer is hundred times lower.For the understanding of translation, i.e. the selection of a specific aminoacyl-tRNA according to codonanticodon recognition, the mechanism of translocation, it is important to know the affinity constants of aminoacyl-tRNA, peptidyl-tRNA and deacylated tRNA towards the A and P sites of the ribosome.It was established earlier in a purely qualitative way that the binding constants with the A site increase in the order deacylated tRNA, peptidyl-tRNA, aminoacyl-tRNA, but decrease in the same order for the P site [I]. Quantitative measurements were realized only in the absence of messenger RNA [2].It was shown in our preceding papers that the codon-dependent binding of tRNA to 30-S subunits is reversible and this enabled us to measure the binding constants of this interaction [3 -61. In the experi-

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“…In this study, we have taken the first steps in this direction by developing and testing a molecular tool that enables spatiotemporal manipulation of collagen synthesis and deposition using light irradiation as remote trigger. We targeted a molecular chaperone, Hsp47 (Heat shock protein 47) that is known to be vital for collagen biosynthesis within cells [15][16][17][18][19] . It is a highly conserved, 47-kDa endoplasmic reticulum (ER)-resident protein known to bind collagen types I to V (and possibly others) 20 , and plays a fundamental role in the folding, stability and intracellular transport of procollagen triple helices 18 .…”

mentioning

confidence: 99%

“…We targeted a molecular chaperone, Hsp47 (Heat shock protein 47) that is known to be vital for collagen biosynthesis within cells [15][16][17][18][19] . It is a highly conserved, 47-kDa endoplasmic reticulum (ER)-resident protein known to bind collagen types I to V (and possibly others) 20 , and plays a fundamental role in the folding, stability and intracellular transport of procollagen triple helices 18 . Procollagen is inherently unstable at physiological temperature and prone to intracellular degradation.…”

mentioning

confidence: 99%

Photoactivatable Hsp47: A Tool to Control and Regulate Collagen Secretion & Assembly

Khan

Sankaran²,

Paez³

et al. 2018

Preprint

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Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. This article reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine -ONBY) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive towards collagen binding. The inactive, photoactivatable protein was easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum (ER) via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turned into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 resulted in stimulated secretion of Collagen. The ability to promote collagen synthesis on-demand, with spatiotemporal resolution, and in disease state cells is demonstrated in vitro. We envision that photoactivatable Hsp47 will allow unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspire new therapeutic concepts in biomedicine and tissue regeneration.

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[9] Conformational analysis of tRNA

Cited by 10 publications

References 8 publications

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

The Mechanism of Codon-Anticodon Interaction in Ribosomes. Heterogeneity of tRNA Complexes with 70-S Ribosomes of Escherichia coli

Photoactivatable Hsp47: A Tool to Control and Regulate Collagen Secretion & Assembly

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[9] Conformational analysis of tRNA

Cited by 10 publications

References 8 publications

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

The Mechanism of Codon-Anticodon Interaction in Ribosomes. Heterogeneity of tRNA Complexes with 70-S Ribosomes of Escherichia coli

Photoactivatable Hsp47: A Tool to Control and Regulate Collagen Secretion &amp; Assembly

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Photoactivatable Hsp47: A Tool to Control and Regulate Collagen Secretion & Assembly