The tpl promoter of Citrobacter freundii is activated by the TyrR protein.

The ability of microorganisms to degrade L-tyrosine to phenol, pyruvate, and ammonia is catalyzed by the inducible enzyme L-tyrosine phenol lyase (EC 4.1.99.2). To investigate possible mechanisms for how the synthesis of this enzyme is regulated, a variety of biochemical and genetic procedures was used to analyze transcription from the tpl promoter of Citrobacter freundii ATCC 29063 (C. braakii). By computer analysis of the region upstream of the tpl structural gene, two segments of DNA bearing strong homology to the known operator targets of the TyrR protein of Escherichia coli were detected. A DNA fragment of 509 bp carrying these operator targets plus the presumptive tpl promoter was synthesized by PCR and used to construct a single-copy tpl-lacZ reporter system. The formation of beta-galactosidase in strains carrying this reporter system, which was measured in E. coli strains of various genotypes, was strongly dependent on the presence of a functional TyrR protein. In strains bearing deletions of the tyrR gene, the formation of beta-galactosidase was reduced by a factor of 10. Several mutationally altered forms of TyrR were deficient in their abilities to activate the tpl promoter. The pattern of loss of activation function was exactly parallel to the effects of the same tyrR mutations on the mtr promoter, which is known to be activated by the TyrR protein. When cells carrying the tpl-lacZ reporter system were grown on glycerol, the levels of beta-galactosidase were 10- to 20-fold higher than those observed in glucose-grown cells. The effect was the same whether or not TyrR-mediated stimulation of the tpl promoter was in effect. By deleting the cya gene, it was shown that the glycerol effect was attributable to stimulation of the tpl promoter by the cyclic AMP (cAMP)-cAMP reporter protein system. A presumptive binding site for this transcription factor was detected just upstream of the -35 recognition hexamer of the tpl promoter. The transcriptional start point of the tpl promoter was determined by chemical procedures. The precise locations of the TyrR binding sites, which were established by DNase I footprinting, agreed with the computer-predicted positions of these regulatory sites. The two TyrR operators, which were centered at coordinates -272.5 and -158.5 with respect to the transcriptional start point, were independently disabled by site-directed mutagenesis. When the upstream operator was altered, activation was completely abolished. When the downstream operator was altered, there was a fourfold reduction in reporter enzyme levels. The tpl system presents a number of intriguing features not previously encountered in TyrR-activated promoters. First among these is the question of how the TyrR protein, bound to widely separated operators, activates the tpl promoter which is also widely separated from the operators.

Coupling of poly(A) site selection and trans-splicing in Leishmania.

Intergenic regions of polycistronic pre-mRNAs of trypanosomatid protozoans are the sites of two processing reactions: polyadenylation of the upstream gene and trans-splicing of the capped miniexon to the downstream gene. Their close proximity and the lack of consensus motifs at poly(A) sites led us to test whether poly(A) site selection is governed by the location of the downstream splice acceptor in the DHFR-TS locus of Leishmania major. Whenever the position of the downstream splice site was altered, the poly(A) site was shifted 400-500 nucleotides upstream of the new splice site. In contrast, when the wild-type poly(A) site was eliminated, the downstream splice site was unaffected, and polyadenylation was maintained 200-500 nucleotides upstream of the splice site. In a second set of experiments, T7 RNA polymerase expressed in Leishmania was used to direct the synthesis of artificial pre-RNAs in vivo whose expression was found to require the presence of a downstream splice acceptor. We conclude that poly(A) site selection in Leishmania is specified by the position of the downstream splice acceptor and propose a scanning model for poly(A) site selection after splice site recognition.