We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV crosslinking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780 -786).Trypanosomes are primitive eukaryotes whose parasitic life cycle involves the differentiation into several successive adaptive forms in different hosts and environments. The main developmental stages are the bloodstream form in the mammalian host and the procyclic form in the tsetse fly vector. Each of these stages is characterized by a major surface protein, the variant surface glycoprotein in the bloodstream form and procyclin in the procyclic form (1, 2). Another major difference between these forms is the energy metabolism. Bloodstream forms possess an inactive mitochondrion and respire through the catabolism of glucose in specialized organelles termed glycosomes, whereas procyclic forms utilize a fully functional mitochondrion for oxidative phosphorylation, and amino acids probably serve as the major carbon source in vivo (3). In the bloodstream, the mitochondrion is reactivated when the trypanosomes differentiate from the proliferative slender form into the quiescent stumpy form through several intermediate stages.The nuclear genome of these organisms appears to be organized in long polycistronic transcription units and probably contains only a few promoters (4 -7). The expression of many genes analyzed so far appears to be stage-specific and reflects the developmental stage of the parasite. Interestingly, genes belonging to the same transcription unit are often differentially stage-regulated, indicating that post-transcriptional processes operating at the levels of RNA maturation, stability, and translation are primarily responsible for controlling cellular differentiation (4 -7). The primary polycistronic transcripts of trypanosomes are rapidly processed into mature mRNAs by trans-splicing and polyadenylation. These processing events appear to be coupled, the choice of a polyadenylation site being apparently dictated by the position of the downstream splice site, probably through the scanning of transcripts by a multifactorial complex enc...

In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.

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The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristics with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational analysis revealed three short critical stretches at positions ؊61 to ؊59 (box 1), ؊38 to ؊35 (box 2), and ؊1 to ؉1 (start site), the spacing of which was essential. These elements were conserved in the promoter for a metacyclic VSG gene. Hybrid sequences containing box 1 of the VSG promoter and box 2 of the ribosomal promoter were active. A specific binding of proteins to the noncoding strand of box 2, but not to double-stranded DNA, occurred. Competition experiments indicated that these proteins also bind to the corresponding region of the metacyclic VSG, procyclin, and ribosomal promoters. Binding of such a protein, of 40 kDa, appeared to be shared by these promoters.The protozoan parasite Trypanosoma brucei shares its life cycle between mammals and an insect vector, the tsetse fly. The bloodstream (mammalian) form and the procyclic (insect) form are uniformly covered by a surface coat, of variant surface glycoprotein (VSG) and procyclin, respectively. The VSG replaces procyclin when procyclic forms differentiate into metacyclic forms in the salivary glands of the fly. This surface antigen persists throughout the development of the parasite in the blood and rapidly disappears when procyclin is reexpressed upon differentiation from the bloodstream to the procyclic form. Therefore, the VSG and procyclin are typical markers for the major developmental stages of the parasite (for recent reviews, see references 4, 10, 24, and 25).African trypanosomes are responsible for important human and animal plagues. They escape the immune defenses of their hosts by a periodical change of their antigenic VSG coat. The study of the control of VSG expression is thus of major fundamental and economic importance. However, the mechanisms controlling gene expression in trypanosomatids are poorly understood. This is particularly true for transcriptional controls. Because of the general organization of genes into polycistronic transcription units, there is only limited information on transcription promoters and their controls. The only promoters for protein-coding genes that are known so far are those of VSG and procyclin (11,12, 23,26,31,32). There are two diploid loci for the procyclin transcription units, and it is believed that the promoters of all these units are simultaneously active in the procyclic form (11,20). The number of VSG transcription units is estimated to be between 6 and 20 (4, 24), but only a single one is expressed at a given time. So far it is not clear if this control operates at the level of promoter activity or transcription attenuation. Interestingly, although the VSG and procyclin units ar...

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Proline cis-trans conformational isomerization is a mechanism that affects different types of protein functions and behaviors. Using analytical characterization, structural analysis, and molecular dynamics simulations, we studied the causes of an aberrant two-peak size-exclusion chromatography profile observed for a trispecific anti-HIV antibody. We found that proline isomerization in the tyrosine-proline-proline (YPP) motif in the heavy chain complementarity-determining region (CDR)3 domain of one of the antibody arms (10e8v4) was a component of this profile. The pH effect on the conformational equilibrium that led to these two populations was presumably caused by a histidine residue (H147) in the light chain that is in direct contact with the YPP motif. Finally, we demonstrated that, due to chemical equilibrium between the cis and trans proline conformers, the antigen-binding potency of the trispecific anti-HIV antibody was not significantly affected in spite of a potential structural clash of 10e8v4 YP trans P trans conformers with the membrane-proximal ectodomain region epitope in the GP41 antigen. Altogether, these results reveal at mechanistic and molecular levels the effect of proline isomerization in the CDR on the antibody binding and analytical profiles, and support further development of the trispecific anti-HIV antibody.

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In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.