In vitro analysis of transcription initiation and termination from the Lhcb1 gene family in Nicotiana sylvestris: detection of transcription termination sites.

Abstract

Genes encoding chlorophyll a/b-binding proteins of photosystem II (Lhcb) constitute a multigene family. Nine Lhcb1 genes have previously been isolated from the tobacco species, Nicotiana sylvestris, and the transcription initiation sites in vivo have been mapped. Reaction conditions from a previously developed in vitro transcription system from tobacco cultured cells were optimized for the Lhcb1 genes. Transcription initiation sites in vitro predominantly coincided with those found in vivo and were typically cytidines, a system unique to N. sylvestris Lhcb1 genes. CTC*A (C* for initiation site) was a consensus motif for the initiation region in vitro, as reported in vivo. Mutation analysis defined functionally that the TATA box is essential for transcription initiation and that the CTCA motif is a determinant of transcription initiation sites but not transcript levels. Polyadenylation sites were determined from in vivo transcripts, located 12-21 nt downstream from likely poly(A) signals. Four major 3'-ends of in vitro transcripts from Lhcb1*6 were detected, 40-300 nt downstream of the poly(A) site, suggestive of multiple, discrete transcription termination sites. These 3'-ends are mapped in or nearby T-rich sequences.