Transcription of aDrosophilatRNAArggene in yeast extract: 5′-flanking sequence dependence for transcription in a heterologous system

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Abstract
The Drosophila tRNA gene encoded on pArg is efficiently transcribed in extracts of Saccharomyces cerevisiae, but the efficiency is 5′-flanking sequence dependent: deletion to between positions −21 and −17 (relative to position +1 of the mature coding sequence) reduces transcription to a very low level. This demonstrates that requirement for wild-type 5′-flanking sequence exists in the case of a heterologous combination of a tRNA gene and transcription extract. Expression of pArg in vivo in S. cerevisiae is also dependent on the wild-type 5′-flanking sequence, but only with deletion to between −17 and −11 is the steady-state level of pArg transcripts reduced to near zero. The 5′-flanking sequence requirement in S. cerevisiae extract is similar to that found in Drosophila Kc cell extract. However, transcription kinetics distinguish S. cerevisiae extract from that of Drosophila Kc cells. tRNA genes added to S. cerevisiae extract exhibit a lag phase before initiation of active transcription, but this lag is much shorter and much less temperature dependent than is the lag phase in Drosophila Kc cell extract.