Several cloned members of the yeast tRNAPhe gene family were transcribed in vitro using a HeLa extract and a yeast extract. The optimum DNA concentration was determined and kinetic experiments were performed for each clone to compare transcription levels. Both extract systems were able to splice the intervening sequence, but only the yeast extract produced the mature product. Some genes were not transcribed with the homologous system while they were transcribed with the HeLa extract, suggesting a control mechanism that is not operating in the heterologous system. Competition experiments demonstrated that the intragenic promoters of the inactive genes were able to bind transcription factor(s), but not as efficiently as active genes. This binding was not so strong when using linear DNA and was dependent on the presence of the 3′ intragenic control region. DNA sequencing and computer analysis indicated the presence of short conserved sequences upstream from the genes. These sequences, which are not related to the intragenic promoters, are direct repeats of part of the 3′ coding region in those genes that are transcribed in the homologous system. The relevance of these sequences on homologous transcription in vitro remains to be established.
ASJC Scopus subject areas
- Molecular Biology