Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Transcriptional specificity after mycobacteriophage I3 infection.

Transcriptional regulation following mycobacteriophage I3 infection has been investigated. For this purpose, RNA polymerase mutants (rifr) of host bacterium, Mycobacterium smegmatis have been isolated and characterised. Phage growth in rifs and rifr cells in presence of rifampicin revealed the involvement of host RNA polymerase in phage genome transcription. This was confirmed by studies on in vivo RNA synthesis as well as by direct RNA polymerase assay after phage infection. Significant stimulation in RNA polymerase activity was seen following phage infection. The maximal levels were attained in about 60 min post infection and maintained throughout the phage development period. The stimulation of polymerase activity was most pronounced when the phage DNA was used as the template. RNA polymerases from uninfected and phage-infected Mycobacterium smegmatis have been purified to homogeniety. The enzyme purification was accomplished by a rapid procedure utilising affinity chromatography on rifampicin-Sepharose columns. Subunit structure analysis of the purified RNA polymerase from uninfected and phageinfected cells showed the presence of α, β, β' and σ subunits similar to the other prokaryotic RNA polymerases. In addition, a polypeptide of 79,000 daltons was associated with the enzyme after phage infection. The enzymes differed in their properties with respect to template specificity. Phage 13 DNA was the preferred template for the modified RNA polymerase isolated from infected cells which may account for the transcriptional switch required for phage development.

The mode of inhibition of the biosynthesis of phenylalanine ammonia lyase by its product cinnamic acid in aging potato parenchyma tissue.

Excised potato parenchyma tissue upon aging in air and light, showed synthesis of RNA, as assessed by the incorporation of [3H]-uridine, reaching a maximum in 18 h. The increase in RNA synthesis was in parallel with the increase in phenylalanine ammonia lyase synthesis. The treatment of the tissue, initially with actinomycin D at 15 μg/g tissue, or with trans-cinnamate at 3·5 mM, caused 65% and 50% inhibition of RNA synthesis ,respectively. The inhibition was reduced to 20% by delaying the treatment of trans-cinnamate to the ninth hour. A comparative study on the nuclear fractions, isolated from trans-cinnamate treated and untreated aged parenchyma tissues showed that trans-cinnamate treatment inhibited the in vitro RNA synthesis by 38%. Studies on the in vivo synthesis of PAL and other proteins showed that trans-cinnamate treatment mainly impaired the de novo synthesis of PAL. The total RNA, isolated from trans-cinnamate treated parenchyma, was 66% less efficient in the translation of PAL, in cell free wheat germ protein synthesizing system. The translation product, purified by affinity chroma tography on phenylalanine conjugated sepharose-4B was found to be homogeneous and showed a single radioactive peak corresponding to protein band on polyacrylamide gel electrophoresis.