Binding of the HIV-1 nucleocapsid protein to the primer tRNA(3Lys), in vitro, is essentially not specific.
The nucleocapsid protein NCp7 of human immunodeficiency virus, type 1, is a key component in the viral life cycle. Since, the first common step of all reported NCp7 activities corresponds to a nucleic acid-binding step, the NCp7 binding parameters to the natural primer tRNA(3Lys) were investigated. Using NCp7 intrinsic fluorescence, we found that (i) in 0.1 M NaCl, NCp7 bound noncooperatively to tRNA(3Lys) with a Kobs = 3.2 x 10(6) M-1 association constant and a n = 6 binding site size, (ii) four ionic interactions were formed in the NCp7.tRNA(3Lys) complex, and (iii) nonelectrostatic factors provided about 60% of the binding energy. These binding parameters were not significantly altered when the natural tRNA(3Lys) was replaced by either an in vitro synthetic tRNA(3Lys) transcript, the heterologous yeast tRNA(Phe) or the structurally unrelated 5 S RNA from Escherichia coli. Moreover, the environment of the intrinsic fluorescent reporters (Trp37 and Trp61) was similar in the various complexes. Finally, experiments performed at low protein concentration provide no evidence of high affinity binding sites. Taken together, our data strongly suggested an essentially nonspecific binding of NCp7 to tRNA(3Lys) and thus did not seem to support a direct role of NCp7, per se, in the selection of tRNA(3Lys) from the pool of cellular tRNAs.
Mély
Y
Y
Laboratoire de Biophysique, URA 491 du CNRS, Université Louis Pasteur de Strasbourg I, Faculté de Pharmacie, Illkirch, France.
de Rocquigny
H
H
Sorinas-Jimeno
M
M
Keith
G
G
Roques
B P
BP
Marquet
R
R
Gérard
D
D
D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism*; D036022:Capsid Proteins; D004926:Escherichia coli; D015683:Gene Products, gag / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012346:RNA, Transfer, Amino Acyl; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015636:Magnesium Chloride