Characterization of a Transcription Terminator of the Procyclin PARP A Unit of Trypanosoma brucei

Abstract

The polycistronic procyclin PARP (for procyclic acidic repetitive protein) A transcription unit of Trypanosoma brucei was completely characterized by the mapping of the termination region. In addition to the tandem of procyclin genes and GRESAG 2.1, this 7.5-to 9.5-kb unit contained another gene for a putative surface protein, termed PAG (for procyclin-associated gene) 3. The terminal 3-kb sequence did not contain significant open reading frames and cross-hybridized with the beginning of one or several transcription units specific to the bloodstream form. At least three separate fragments from the terminal region were able to inhibit chloramphenicol acetyltransferase expression when inserted between either the PARP, the ribosomal, or the variable surface glycoprotein promoter and a chloramphenicol acetyltransferase reporter gene. This inhibition was due to an orientation-dependent transcription termination caused by the combination of several attenuator elements with no obvious sequence conservation. The procyclin transcription terminator appeared unable to inhibit transcription by polymerase II.In Trypanosoma brucei, the genes seem to be generally organized in polycistronic transcription units (8,21). Because of this particular organization, only two trypanosomal promoters for protein-encoding genes are known, namely, those for the major stage-specific surface antigens, the variant surface glycoprotein (VSG) and procyclin (9,12,23,29,32). These promoters may recruit an RNA polymerase of the ribosomal type, although this remains equivocal (7,25). To date, no DNA sequence able to terminate transcription has been found in these organisms.Procyclin is the major surface glycoprotein of the procyclic form of T. brucei (27). The procyclin genes are organized in tandem pairs present in two diploid loci termed PARP (for procyclic acidic repetitive protein), A and PARP B, with the possible presence of an additional gene copy in some trypanosome strains (9,14,29). The transcription promoter is present immediately upstream from the first gene copy of each locus, and it seems that the different loci are transcribed simultaneously (9,14,29). In each locus, transcription is polycistronic, with at least one gene associated with the tandem of procyclin genes, GRESAG 2.1 in PARP A and PAG (for procyclinassociated gene) 1 in PARP B (2, 15). The approximative extent of each transcription unit was estimated to be less than 10 kb, contrasting with the larger size of units for other genes (2,9,15,25,29).In this paper, we report the complete characterization, from the promoter to the termination region, of the PARP A unit. There appear to be no more than four genes in this unit. Interestingly, the 3Ј-end region of the PARP A unit was found to cross-hybridize with the 5Ј-end region of at least one bloodstream-specific transcription unit. The termination region appeared to extend over 2 kb. Different fragments from this region efficiently inhibited chloramphenicol acetyltransferase (CAT) expression driven by either the PARP, the ribosom...

Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids

“…In L. tarentolae, SL RNA is most probably transcribed by pol II (62). Most aspects of pol II transcription in trypanosomatids are poorly understood, and little is known about how pol II transcription terminates in trypanosomatids (6,39) and other organisms (64). However, termination of small RNA gene transcription is controlled by a well-defined 3Ј box (64).…”

MicroRNAs of parasites: current status and future perspectives

“…Furthermore, a number of miRNA hairpins have been found in clusters of multiple identical copies. The target proteins, 20S proteosome, GM6, and GRESAG 4.2 corresponding to these clustered miRNAs, play essential role in trypanosomiasis (Berberof et al 1996;To and Wang 1997). These miRNAs can act as genetic switches modulating host-parasite interaction and provide useful clue toward control of trypanosomiasis (Mallick et al 2008).…”

Overlapping sense and antisense transcription units in Trypanosoma brucei

“…There are several expression site‐associated genes ( ESAG s; reviewed by Pays and Nolan, 1998) between the promoter and the VSG gene, which is the last gene in the transcription unit. In contrast, the procyclin expression sites are short transcription units, previously estimated to be 6–9 kb in length (Rudenko et al ., 1990; Berberof et al ., 1991; 1996; Koenig‐Martin et al ., 1992), that are located internally on chromosomes VI and X (Melville et al ., 1998). Each expression site starts with two or three procyclin genes followed by a procyclin‐associated gene ( PAG; reviewed by Roditi et al ., 1998).…”

“…The ends of these transcription units have not been determined. Sequences have been identified that attenuate transcription from the GPEET/PAG3 expression site on chromosome VI (Berberof et al ., 1996); these appear to bind a protein factor (Berberof et al ., 2000). A gene encoding a microtubule‐associated repetitive protein (MARP) is part of a Pol II transcription unit upstream of the EP/PAG1 expression site (Koenig et al ., 1989), but no Pol II attenuator or terminator sequences have been identified between the MARP gene and the procyclin promoter.…”