Sequence at the Site of Attachment of an Affinity‐Label Derivative of Puromycin on 23‐S Ribosomal RNA of Escherichia coli Ribosomes

Abstract

The puromycin analogue, 5′O‐(N‐bromoacetyl‐p‐aminophenylphosphoryl)‐3′‐N‐L‐phenylalanyl puromycin aminonucleoside, acts as an affinity label in covalently binding to 23‐S RNA in the active centre of peptidyl transferase of Escherichia coli ribosomes [Greenwell, P., Harris, R. J. & Symons, R. H. (1974) Eur. J. Biochem. 49, 539–554]. The sequence of a pentanucleotide fragment of 23‐S RNA to which the affinity label is attached has now been determined.In an attempt to determine the nucleotide residue to which the affinity label was attached, a 32P‐labelled affinity label was reacted with ribosomal RNA, poly(A), poly(C), poly(G) and poly(U) under the conditions used for affinity labelling of ribosomes. However, no specific reaction occurred, confirming our earlier observation that this required the functional integrity of the ribosome. An alternative approach of affinity labelling ribosomes labelled by incubating E. coli in the presence of [3H]adenosine, [3H]cytidine, [3H]guanosine or [3H]uridine, showed that the affinity label was attached to a CMP residue.Affinity‐labelled oligonucleotide fragments were purified for sequence analysis as follows. The free α‐amino group of the affinity‐label molecule attached to 23‐S ribosomal [32P]RNA was coupled to biotin by reaction with the N‐hydroxysuccinimide ester of biotin. After digestion of the RNA with either ribonuclease A or T1, the basic protein avidin was added to form the avidin · biotin – affinity‐label‐oligonucleotide fragment. Stepwise chromatography on phosphocellulose, followed (after dissociation of the avidin) by electrophoresis on DEAE‐cellulose at pH 1.9 gave substantial purification of the biotin–affinity‐label‐oligonucleotide. From the nucleotide composition of the purified ribonuclease A and T1 fragments, the pentanucleotide sequence of G‐U‐C*‐C‐Gp was obtained, where C* represents C modified by the affinity label.