The RNA-binding domain of transcription termination factor rho: isolation, characterization, and determination of sequence limits.
The function of transcription termination factor rho from Escherichia coli is dependent upon its ability to bind RNA. To delineate the extent of the RNA-binding domain in the rho polypeptide, plasmid-borne copies of altered forms of the rho gene were expressed to yield truncated versions. These proteins were then isolated and assayed for their ability to bind an RNA oligonucleotide [oligo(C)8] using an ultraviolet light-induced cross-linking assay. A fragment consisting of the first 116 amino acid residues, rho(1-116), bound oligo(C)8 with nearly the same affinity and specificity as the intact protein. Smaller derivatives lacking 5, 13, or 22 residues from the N terminus or with 2 fewer residues at the C terminus bound RNA with reduced affinity, while derivatives lacking 27 N-terminal residues or having just the first 109 residues were unable to bind RNA. Derivatives lacking N-terminal residues were considerably less soluble than rho(1-116). The physical properties of rho(1-116) indicate that it possesses approximately 20% each of alpha-helix and beta-sheet and is monomeric in solution. Thus, the results show that this fragment, which contains an RNP1 sequence motif, will be a good model for future physical-chemical studies of the protein-RNA interactions of rho.
Modrak
D
D
Department of Chemistry, Indiana University, Bloomington 47405.
Richardson
J P
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000737:chemistry; D017353:Gene Deletion; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D012234:Rho Factor; D012313:RNA