Role of a bulged A residue in a specific RNA-protein interaction.
The translational operator of the R17 replicase gene contains a bulged A residue that is essential for the specific binding to R17 coat protein. A large number of operator variants have been synthesized to more precisely examine the role of the bulged A residue on this specific protein-RNA interaction. By use of RNA ligase and transcription of synthetic DNA templates by T7 RNA polymerase, 14 different nucleotides were introduced to the bulged A position of three different coat protein binding fragments. The affinity between coat protein and each fragment was determined by a nitrocellulose filter binding assay. The data indicate that while functional groups on N1, C2, C6, N7, and 2'OH of the bulged A can be substituted without greatly changing protein binding, bulky substituents cannot be tolerated at these positions. Data from additional fragments that have base-pair changes adjacent to the bulged A suggest that the propensity of the bulged A to intercalate into the helix can affect protein binding.
Wu
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HN
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Uhlenbeck
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OC
D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D007202:Indicators and Reagents; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000378:metabolism*
D004279:DNA, Viral; D007202:Indicators and Reagents; D009843:Oligoribonucleotides; D014759:Viral Envelope Proteins; D011130:RNA Ligase (ATP)