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The RNA-binding domain of transcription termination factor rho: isolation, characterization, and determination of sequence limits

Overview of Modrak D et al.

AuthorsModrak D  Richardson JP  
AffiliationDepartment of Chemistry   Indiana University   Bloomington 47405.  
JournalBiochemistry
Year 1994

Abstract


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.