Das Problem der heterologen Ablesung

Initiation complexes formed by E. coli ribosomes in the presence of 32P-labeled A protein initiator region from R17 bacteriophage Rna have been treated with colicin E3 and disassembled by exposure to 1% sodium dodecyl sulfate. Electrophoresis on 9% polyacrylamide gels reveals a dissociable complex containing the 30-nucleotide-long messenger fragment and the 50-nucleotide-long colicin fragment, which arises from the 3' terminus of the 16S RNA. The complex is a pure RNA-RNA hybird; it is apparently maintained by a seven-base complementarity between the two RNA fragments. Detection of this mRNA-rRNA complex strongly supports the hypothesis that during the initiation step of protein biosynthesis the 3' end of 16S RNA base pairs with the polypurine stretch common to initiator regions in E. coli and bacteriophage mRNAs. The implications of our findings with respect to the molecular mechanism of initiation site selection and mRNA binding to ribosomes, the role of rRNA in ribosome function, and species specificity in translation are explored.

RNA.DNA hybridization experiments utilizing separated strands of HeLa mitochondrial DNA and mit-RNA from HeLa cells exposed to short pulses of [5-(3)H]uridine have shown that the labeled RNA hybridizes with both the light (L) and the heavy (H) strand, though to a different relative extent depending upon the labeling time. Thus, hybridization of pulse-labeled RNA is about equal with the two strands when the pulse is very short (1-5 min), and becomes more and more predominant with the H strand with increasing pulse length. Pulse-labeled fast-sedimenting mit-RNA forms RNase-resistant double-stranded structures up to more than 5 mum long when self-annealed or annealed with an excess of unlabeled mit-RNA. These observations and the previous evidence of complete transcription of the H strand strongly suggest that mit-DNA is transcribed in HeLa cells symmetrically over a considerable portion of its length, with the transcript of the L strand being rapidly degraded or otherwise removed from the mitochondrial fraction.