Synthesis of Biologically Active Portions of an Intercistronic Region by Use of a New 3′‐Phosphate Incorporation Method to Protect 3′‐OH and Their Binding to Ribosomes

Abstract

To examine the influence of bases contiguous to a starter codon, a rapid means of assembling biologically active regions corresponding to portions, or to analogues of portions of intercistronic regions is desirable. To do this, a chemical method for the specific insertion of 3′‐monophosphate groups on to chemically synthesized trinucleotides, tetranucleotides and hexanucleotides of defined sequence bearing different bases at the 3′ terminus has been devised. The method involved phosphorylation of blocked oligoribonucleotides synthesized by a phosphotriester method. The deblocked oligoribonucleotides were phosphorylated at the 5′ end with T4‐induced polynucleotide kinase. The products of this kinase reaction served as“donors” in RNA ligase reactions. The [32P]pC‐A‐U‐A‐U‐Gp, [32P]pA‐U‐Gp, [32P]pU‐A‐A, [32P]pA‐G‐G‐Ap, [32P]pC‐U‐U‐Ap and [32P]pU‐C‐C‐Up “donors” were used to synthesize A‐G‐G‐A[32P]pC‐A‐U‐A‐U‐Gp, U‐C‐C‐U[32P]pC‐A‐U‐A‐U‐Gp, U‐A‐A[32P]pA‐U‐G, A‐U‐G[32P]pU‐A‐A, U‐A‐A‐G[32P]pA‐G‐G‐Ap, U‐C‐C‐U[32P]pC‐U‐U‐Ap and A‐U‐U‐C[32P]pU‐C‐C‐Up indicating that the method functions with all bases. A‐U‐Gp, PA‐U‐Gp and pC‐A‐U‐A‐U‐Gp were isolated free of reactants and, along with PA‐U‐G, were all shown to promote the formation of translational initiation complexes. A‐G‐G‐A[32P]pC‐A‐U‐A‐U‐Gp, which corresponds to the 5′‐terminal portion of the intracistronic region of the maturation protein of bacteriophage Qβ, bound more efficiently to Escherichia coli ribosomes than the U‐C‐C‐U[32P]pC‐A‐U‐A‐U‐Gp, [32P]pC‐A‐U‐A‐U‐Gp, [32P]pA‐U‐G‐U‐A‐A or [32P]pU‐A‐A‐A‐U‐G controls. The results suggested that the content and number of residues at the 5′ terminus attached to A‐U‐G affect binding of oligonucleotides to ribosomes; purine nucleosides appear to be more effective than pyrimidine nucleosides in this regard.

The Oxazolidinone Linezolid Inhibits Initiation of Protein Synthesis in Bacteria

“…), and [ 35 S]tRNA fMet was synthesized as described by Ganoza et al (9). AUG was synthesized by the method of Nielson et al (20). The oligoribonucleotide used in the S. aureus initiation complex assays had the sequence 5Ј-rGGGAAUUCGGAGGUUUAAAAAUGGGUAAA-3Ј and was purchased from Integrated DNA Technologies (Coralville, Iowa).…”