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Nucleic Acids Research, 1995, Vol. 23, No. 15 2914-2918
© 1995

MOLECULAR BIOLOGY

Mutational analysis of the transcription start site of the yeast tRNALeu3 gene

Paolo Fruscoloni1,2, Michela Zamboni3, Gianna Panetta1, Augusto De Paolis and Glauco P. Tocchini-Valentinl1,2,*

1Institut of Cell Biology CNR, Viale Mark 43, 00137 Rome, Italy 2Department of Biochemistry and Molecular Biology, University of Chicago Chicago, IL 60637, USA 3Enichem, Istituto Donegani 0005 Monterotondo, Rome, Italy

*To Whom correspondence should be addressed at: Institute of Cell biology, CNR, Viale Marx 43, 0037 Rome, Italy

Received April 20, 1995. Accepted June 20, 1995.

In addition to the well-known internal promoter elements of tRNA genes, 5' flanking sequences can also influence the efficiency of transcription by Saccharomyces cerevlslae extracts In vitro. A consensus sequence of yeast tRNA genes in the vicinity of the transcriptional start site can be derived. To determine whether the activity of this region can be attributed to particular sequence features we studied In vitro mutants of the start site region. We found that the start site can be shifted, but only to a limited extent, by moving the conserved sequence element. We found that both a pyrimidine-purlne motif (with transcription initiating at the purine) and a small T: A base pair block upstream are important for efficient transcription In vitro. Thus the sequence surrounding the start site of transcription of the yeast tRNALeu3 gene does play a role In determining transcription efficiency and fixing the precise site of initiation by RNA polymerase III.


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