5,6‐Dichlororibofuranosylbenzimidazole Inhibits the Rate of Transcription Initiation in Intact Chironomus Cells

Open Access
Publisher Website
Google Scholar
Abstract
The effect of nucleoside analogue 5.6‐dichloro‐l‐β‐D‐ribofuranosylbenzimidazole on RNA transcription has been reinvestigated after the observation that explanted salivary gland cells of Chironomus tentans efficiently take up of exogeneous ATP in an undegraded form. We addressed the question of whether the inhibitory action is exerted predominantly by accentuation of premature termination of hnRNA transcripts or by inhibition of the initiation step. Electrophoretic analyses in 10% polyacrylamide gels of [α‐32P]ATP‐labelled low‐molecular‐weight RNA chains in the 30–1000‐nucleotide range did not reveal the accumulation of early terminated transcripts irrespective of whether dichlororibosylbenzimidazole was present in the medium or not. The separation of 70% ethanol‐soluble α‐32P‐labelled material by thin‐layer electrophoresis led to a similar result: no detectable accumulation of very short transcripts (oligonucleotides) due to a possible premature termination of transcription. A selective labelling of newly initiated RNA transcripts was achieved by incorporation of 32P from [β‐32P]ATP into the 5′ termini of RNA molecules which start with ATP.The internal uptake of 32P was low, if any, as measured by the sensitivity of RNA labelling aganist alkaline phosphatase. In addition to a significant portion of β‐32P‐labelled hnRNA chains, labelling of newly initiated preribosomal 38‐S RNA, and 75‐S RNA of Balbiani ring origin, is demonstrated as well. Incorporation of label into 38‐S and 75‐S RNA exhibits the same kind of specificity against dichlororibosylbenzimidazole as when isotopic nucleosides are used for RNA labelling: i.e. the formation of labelled 38‐S RNA is resistant while that of 75‐S RNA is sensitive. During a 5‐min pulse, in the presence of the analogue, the rate of accumulation of newly initiated β‐32P‐labelled transcripts, predominantly hnRNA, was inhibited by more than 60%. The data taken together strongly indicate that the inhibition of the transcription of hnRNA genes with 5,6‐dichloro‐ ribofuranosylbenzimidazole in Chironomus takes place at the level of initiation and argues against the existence of prematurely terminated hnRNA transcripts