Specificity of phage display selected peptides for modified anticodon stem and loop domains of tRNA.
Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.
Eshete
Matthewos
M
Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Campus Box 7622, Raleigh, NC, 27695-7622, USA.
Marchbank
Marie T
MT
Deutscher
Susan L
SL
Sproat
Brian
B
Leszczynska
Grazyna
G
Malkiewicz
Andrzej
A
Agris
Paul F
PF
D020816:Amino Acid Motifs; D000926:Anticodon / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020029:Base Pairing; D003062:Codon / Q000737:chemistry; D006801:Humans; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011560:Pseudouridine / Q000737:chemistry; D012352:RNA, Transfer, Glu / Q000737:chemistry*; D012357:RNA, Transfer, Lys / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000737:chemistry
D000926:Anticodon; D003062:Codon; D019151:Peptide Library; D010455:Peptides; D012352:RNA, Transfer, Glu; D012357:RNA, Transfer, Lys; D013891:Thiouridine; D011560:Pseudouridine