Functional Recognition of the Modified Human tRNA^Lys3_UUU Anticodon Domain by HIV's Nucleocapsid Protein and a Peptide Mimic

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• 作者：William D. Graham¹ ; ^† ; ; Lise Barley-Maloney¹ ; ¹ ; ^† ; ; ^{Lise_Barley-Maloney@bd.com"" rel=""nofollow} ; Caren J. Stark² ; ^† ; ; Amarpreet Kaur¹ ; Khrystyna Stolyarchuk¹ ; Brian Sproat³ ; ¹ ; ^{fa205401@skynet.be"" rel=""nofollow} ; Grazyna Leszczynska⁴ ; Andrzej Malkiewicz⁴ ; Nedal Safwat¹ ; ¹ ; ^{nedal.safwat@biomerieux.com"" rel=""nofollow} ; Piotr Mucha⁵ ; Richard Guenther¹ ; ¹ ; ^{Dick_Guenther@ncsu.edu"" rel=""nofollow} ; Paul F. Agris² ; ¹ ; ^{PAgris@albany.edu"" rel=""nofollow}
• 关键词：Modified nucleosides ; N⁶-threonylcarbamoyladenosine ; 5-methoxycarbonylmethyl-2-thiouridine ; NCp7 ; anticodon stem and loop
• 刊名：Journal of Molecular Biology
• 出版年：2011
• 出版时间：22 July 2011
• 年：2011
• 卷：410
• 期：4
• 页码：698-715
• 全文大小：1516 K

文摘

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA^Lys3_UUU as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA^Lys3_UUU, the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL^Lys3) were studied. NCp7 bound the hASL^Lys3_UUU modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm⁵s²U₃₄) and 2-methylthio-N⁶-threonylcarbamoyladenosine at position-37 (ms²t⁶A₃₇) with a considerably higher affinity than the unmodified hASL^Lys3_UUU (K_d = 0.28 ± 0.03 and 2.30 ± 0.62 μM, respectively). NCp7 denatured the structure of the hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉ more effectively than that of the unmodified hASL^Lys3_UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K_d = 0.55 ± 0.10 μM) and specificity for the ASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇ comparable to that of NCp7. The peptides recognized a t⁶A₃₇-modified ASL with an affinity (K_d = 0.60 ± 0.09 μM) comparable to that for hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇, indicating a preference for the t⁶A₃₇ modification. Significantly, one of the peptides was capable of relaxing the hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉ structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA^Lys3_UUU have been found to be important determinants of NCp7's recognition prior to the tRNA^Lys3_UUU being annealed to the viral genome as the primer of reverse transcription.