24] Analysis of modified nucleosides and nucleotide sequence of tRNA

Abstract

This chapter discusses the modified nucleosides and nucleotide sequence of transfer ribonucleic acid (tRNA). The outline of the procedure for sequencing tRNA by the postlabeling method is illustrated in the chapter, which consists of five steps. The chapter describes a sensitive method for the detection of a new modified nucleoside in tRNA. By the method, a sample of 2.5 μg of unfractionated tRNA is sufficient for analysis of modified nucleosides, and the location of these modified nucleosides in the tRNA molecule can also be estimated. In the method, RNA fragments containing a labeled nucleotide at their 5' end are fractionated on a denaturing polyacrylamide slab gel before the analysis of labeled compounds. Therefore, labeled contaminants present in the [γ-32p] adenosine triphosphate (ATP) used for postlabeling do not interfere with the analysis of labeled nucleotides by thin-layer chromatography. tRNAs such as tRNA ser and tRNA TM have a long extra arm, which is about 10 nucleotides longer than those of other tRNAs. These tRNAs possibly constitute only about 5% of the total tRNA. The method described in the chapter is generally useful for tRNAs with a short extra arm. However, the efficiency of phosphorylation of modified nucleosides by polynucleotide kinase treatment with [γ-32p] ATP may not be constant. Therefore, when an unusual modified nucleoside is found by this method, its exact location must be confirmed by total nucleotide sequence determination using a purified sample of the tRNA species containing this nucleoside. The purity of tRNA is the most important factor for nucleotide sequence analysis by the postlabeling technique as described in the chapter.