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Volumn 273, Issue 5281, 1996, Pages 1547-1551

α Helix-RNA major groove recognition in an HIV-1 Rev peptide-RRE RNA complex

Author keywords

[No Author keywords available]

Indexed keywords

ASPARAGINE; RNA; VIRUS DNA;

EID: 0029784592     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5281.1547     Document Type: Article
Times cited : (551)

References (51)
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    • note
    • 15N-ammonium sulfate (0.7 g/liter), or both (Cambridge Isotope Laboratories). Peptides were isolated and purified as described (25). Yields of peptide per liter of culture were ∼10 mg and ∼4 mg for unlabeled and labeled peptide, respectively.
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    • 4 (pH 6.5), 100 mM NaF, 5°C] (26), compared with ∼50% for the modified synthetic peptide (3). Despite the lower α-helical content, the expressed peptide retains specific RNA-binding activity and gives RNA chemical shifts and NOEs identical to a complex with the synthetic peptide (7).
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    • note
    • -2, respectively No nonexperimental torsion restraints were used to enforce A-form geometry for any region of the RNA. A quartic repulsive function was used for nonbonding contacts in all calculations, and electrostatic interactions were completely neglected. A total of 140 distance geometry structures were generated and input into a high-temperature (1000 K) simulated-annealing protocol using the AMBER forcefield with the masses for all the atoms set to 100 [A. T. Brunger, X-PLOR User Manual, Version 3.1 (Yale Univ. Press, New Haven, CT, 1992)]. The force constants for the covalent geometry, distance and dihedral, and nonbonded terms were sequentially scaled from 1% to full value over 36 ps of dynamics in 3-fs steps. The temperature was then cooled to 10 K over 21 ps. The 30 structures with the lowest NMR restraint violation energies were chosen for further refinement. Peptide hydrogen bonds were added for the well-defined α helix at this stage. The refinement protocol consisted of 6 ps of dynamics (1-fs steps) at 500 K, scaling the nonbonded terms from 10% to full value. The temperature was then exponentially coded to 10 K over 9 ps, followed by 100 and 500 steps of steepest and conjugate gradient minimization, respectively. The 19 structures with the lowest restraint violation energies and no violations >0.3 Å were chosen for detailed structural analysis. An average structure (SA) was generated by superimposing the coordinates of all 19 structures and then averaging the coordinates. The covalent geometry of the average structure was regularized by 100 steps of steepest descent and 500 steps of conjugate gradient minimization.
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    • The major groove width of the RNA was quantitated by measuring the shortest phosphorus atom distances across the groove and subtracting 5.8 Å for the van der Waals radii of the phosphate group
    • The major groove width of the RNA was quantitated by measuring the shortest phosphorus atom distances across the groove and subtracting 5.8 Å for the van der Waals radii of the phosphate group.
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    • note
    • We thank J. Puglisi for helpful discussions, C. Turner and A. Brodsky for assistance with NMR experiments, M. Milholten and P. Kim for providing the pMM and pTM plasmids, M. Oakley and C. Cilley for assistance in purification of peptides, and C. Pabo for critical comments. J.L.B. was supported in part by the NIH Inter-departmental Biotechnology Program (GM-08344), and R.T. is a Scholar for the American Foundation for AIDS Research (70405-15-RF). Supported by grants from the National Sciences and Engineering Research Council of Canada (L.E.K.), the Medical Research Council of Canada (L.E.K.), the Searle Scholars Program of the Chicago Community Trust (J.R.W.), and NIH grants GM-39589 (A.D.F. and J.R.W.) and GM-53320 (J.R.W.).


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