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The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression.

ABSTRACT: We have previously reported the co-purification of a tRNA-like molecule with the Trypanosoma brucei SRP complex [Béjà et al . (1993) Mol. Biochem. Parasitol . 57, 223-230]. To examine whether the trypanosome SRP has a unique composition compared with that of other eukaryotes, we analyzed the 7SL RNA and the SRP complex of the monogenetic trypanosomatid Leptomonas collosoma. The 7SL RNA from L. collosoma was cloned, and its gene was sequenced. In contrast to T. brucei , two 7SL RNA transcripts were detected in L.collosoma that originate from a single-copy gene. Using stable cell lines expressing tagged 7SL RNA, we demonstrate that the tRNAArggene located 98 bp upstream to the 7SL RNA serves as part of the 7SL RNA extragenic promoter. The steady-state level of 7SL RNA was found to be tightly regulated, since the presence of the gene on the multi-copy plasmid repressed the synthesis of the chromosomal gene. Cell lines carrying truncated 7SL RNA genes were established and their expression indicated that domain I is essential for expressing the 7SL RNA. No constructs carrying portions of the 7SL RNA were expressed, except for a construct which lacked 23 nt from the 3'end of the RNA. This suggests that 90% of the 7SL RNA molecule is important for its maintenance as a stable small RNA. We propose that the repression phenomenon may originate from a regulatory mechanism that coordinates the level of the 7SL RNA by its binding proteins.

SUBMITTER: Ben-Shlomo H

PROVIDER: S-EPMC147140 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression.

Ben-Shlomo H H Levitan A A Béjà O O Michaeli S S

Nucleic acids research 19971201 24

We have previously reported the co-purification of a tRNA-like molecule with the Trypanosoma brucei SRP complex [Béjà et al . (1993) Mol. Biochem. Parasitol . 57, 223-230]. To examine whether the trypanosome SRP has a unique composition compared with that of other eukaryotes, we analyzed the 7SL RNA and the SRP complex of the monogenetic trypanosomatid Leptomonas collosoma. The 7SL RNA from L. collosoma was cloned, and its gene was sequenced. In contrast to T. brucei , two 7SL RNA transcripts we ...[more]

PMID: 9396805

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Project description:A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native P phaC , with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase gene ispS led to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necatorIMPORTANCE This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and two negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

Project description:BackgroundThe protozoan pathogens Leishmania major, Trypanosoma brucei and Trypanosoma cruzi (the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an in silico analysis of the Tritryps genome sequences.ResultsOur analysis indicated the presence of 83, 66 and 120 genes in L. major, T. brucei and T. cruzi, respectively. These numbers include several previously unannotated selenocysteine (Sec) tRNA genes. Most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes. The distribution of genes in the L. major genome does not seem to be totally random, like in most organisms. While the majority of the tRNA clusters do not show synteny (conservation of gene order) between the Tritryps, a cluster of 13 Pol III genes that is highly syntenic was identified. We have determined consensus sequences for the putative promoter regions (Boxes A and B) of the Tritryps tRNA genes, and specific changes were found in tRNA-Sec genes. Analysis of transcription termination signals of the tRNAs (clusters of Ts) showed differences between T. cruzi and the other two species. We have also identified several tRNA isodecoder genes (having the same anticodon, but different sequences elsewhere in the tRNA body) in the Tritryps.ConclusionA low number of tRNA genes is present in Tritryps. The overall weak synteny that they show indicates a reduced importance of genome location of Pol III genes compared to protein-coding genes. The fact that some of the differences between isodecoder genes occur in the internal promoter elements suggests that differential control of the expression of some isoacceptor tRNA genes in Tritryps is possible. The special characteristics found in Boxes A and B from tRNA-Sec genes from Tritryps indicate that the mechanisms that regulate their transcription might be different from those of other tRNA genes.

Dataset Information

The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression.

Publications

The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression.

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