Effects of rho-independent terminators on the expression of the prokaryoticβ-glucuronidase gene in tobacco

Abstract

The threonine (thr) attenuator with a dyad symmetrical structure is from the regulatory region of the thr operon of Escherichia coli, and encodes RNA with a stem-and-loop structure followed by a stretch of uridine residues. The thr attenuator and its variants were subcloned into the region between the 35S promoter and β-glucuronidase (GUS) coding region, and the transient expression of GUS gene in tobacco protoplast was treated as a reporter for gene regulation in plants. Results from the 14 variants in the stem region of the thr attenuator indicated that both base pairing and sequence specificity in the G+C-rich region of the stem were important for the GUS expression, but 1 base mismatch in the A+U-rich region of stem did not affect the GUS expression in plants. Seven variants with nested deletion in the stretch of uridine residues were also analyzed, and the results suggested that the variants with the shorter uridine stretch produced more GUS protein than those with the longer stretch. Transgenic tobacco plants with the thr attenuator and its variants located between the 35S promoter and GUS coding region were also generated, and their steady state RNAs were hybridized with 2 radioactive antisense RNA probes which bound 5′ and 3′ of the thr hairpin, respectively. After the digestion of S1 nuclease, the amount of the nuclease-resistant transcript from the protection of the 5′ antisense RNA probe was much more than that from the protection of the 3′ probe in all tested variants. This result suggests that these dyad symmetries may affect transcription of plant RNA polymerase II.