Fig. 7.

The 5.5 protein copurifies with tRNA. (A) After purification by gel filtration chromatography, associated nucleic acid was removed by phenol-choloroform extraction (5.5 preparation) and subsequently treated with DNase I or RNaseA as indicated. The plasmid control shown in the final two lanes demonstrates the activity of the DNase I used in the assay. (B) End labeling of isolated RNA reveals that it is heterogeneous and labels poorly at its 5′ end. Copurifying low-molecular-weight RNA was end labeled at the 3′ end with [³²P]pCp and RNA ligase or at the 5′ end with [γ-³²P]ATP using T4 polynucleotide kinase. The bar adjacent to the autoradiogram indicates the low-abundance (as determined by Sybr stain), presumably degraded, RNA that was efficiently labeled by PNK. (C) Diagram of tRNA^Asp showing, boxed and in bold, the segment obtained by cloning after reverse transcription of in vitro-polyadenylated RNA. Similar truncated clones were obtained from tRNA^Ala and tRNA^Thr. EthBr, ethidium bromide; nt, nucleotides.