A technique for mapping transfer RNA genes by electron microscopy of hybrids of ferritin-labeled transfer RNA and DNA: The- φ80hp su +,−III system

Abstract

A method for mapping transfer RNA genes on single strands of DNA is described. tRNA is covalently coupled to the electron-opaque label, ferritin. The ferritinlabeled tRNA, Fer-tRNA, is hybridized to a single strand of DNA, or to a single- strand region of a DNA in a heteroduplex. The sites where the Fer-RNA binds to the complementary sequence on the DNA are then mapped by electron microscopy. Several alternative coupling procedures are described (see Fig. 1). In HzI a — COCH 2Br group is attached to ferritin by acylation. 3'-Oxidized tRNA is joined to HSRCONHNH 2 by hydrazone formation. Ferritin is then coupled to tRNA by reaction of the CBr and SH bonds. In the BI procedure a lysine amino group of ferritin is coupled by Schiff base formation with 3'-oxidized RNA. The conjugate is stabilized by borohydride reduction. In the BII procedure, a —COCH 2Br group is attached to ferritin. (H 2NCH 2CH 2S—) 2 is coupled to oxidized tRNA by Schiff base formation and borohydride reduction. An SH group is exposed by reduction. This HS-tRNA is coupled to a —COCH 2Br group attached to ferritin. All the procedures work but BII is recommended. Methods for purifying the Fer-tRNA and the Fer-tRNA-DNA hybrid are described. For the transducing phages, φ80hp su +,− III and φ80hp su − III, the DNA molecules each carry a piece of bacterial DNA of length 0·066±0·007 λ unit (3100 nucleotide pairs; we find the length of λ is 8·99 φX174 units) replacing a piece of phage DNA of φ80h of length 0·045±0·005 λ unit. The left junction of this bacterial DNA with phage DNA (referred to as P-B′) is at or close to the att site. The two tandem tyrosine genes of φ80hp su +,− III and the single tRNA gene of φ80hp su − III have been mapped at a position 1100 nucleotides to the right of the left ( P· B′) junction of phage DNA and bacterial DNA, by hybridizing Escherichia coli Fer-tRNA to φ80hp su III/φ80h heteroduplexes. The separation of the two ferritin labels in φ80hp su +,− III hybrids gives 140±20 nucleotides as the size of a single tyrosine tRNA gene.