Role of a bulged A residue in a specific RNA-protein interaction

Biochemistry. 1987 Dec 15;26(25):8221-7. doi: 10.1021/bi00399a030.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA, Viral / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Genes*
  • Genes, Viral*
  • Indicators and Reagents
  • Nucleic Acid Conformation
  • Oligoribonucleotides / chemical synthesis*
  • RNA Ligase (ATP) / genetics
  • RNA Ligase (ATP) / metabolism
  • T-Phages / enzymology*
  • T-Phages / genetics
  • Templates, Genetic
  • Transcription, Genetic
  • Viral Envelope Proteins / metabolism*

Substances

  • DNA, Viral
  • Indicators and Reagents
  • Oligoribonucleotides
  • Viral Envelope Proteins
  • RNA Ligase (ATP)