Specific Binding of Proteins to the Noncoding Strand of a Crucial Element of the Variant Surface Glycoprotein, Procyclin, and Ribosomal Promoters of Trypanosoma brucei

Abstract

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...

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…The very simple structure and ribosomal nature of the ES promoters (39,40) is in keeping with the absence of regulatory sequences. In support of this notion, promoters from active or inactive ESs share the same sensitivity to DNase (24), and antigenic variation still occurs properly if the promoter of the active ES is replaced by the ribosomal promoter (41,42). Other arguments include the detection of transcripts originating from inactive ESs such as RNA transcribed from the ES beginning in procyclic forms (29), RNA from antibiotic-resistance genes inserted in inactive ESs (25), or RNA from different ESAG6 from inactive ESs, accounting for up to 20% of the total ESAG6 transcripts (28).…”

Ex Vivo and In Vitro Identification of a Consensus Promoter for VSG Genes Expressed by Metacyclic-Stage Trypanosomes in the Tsetse Fly

“…BES promoters, although highly conserved at the sequence level, require only two elements, boxes 1 and 2, for transcriptional activity (30,37). Both boxes are required to form a stable DNA-protein complex in electrophoretic mobility shift assays or to compete with a specific in vitro transcription signal (22,31), which suggests that they cooperate in the recruitment of trans-acting factors.…”

“…Vanhamme and colleagues (37), by testing for putative box 2 activity in the possible 11.17 MVSG promoter, also showed that the region corresponding to Ϫ36 to Ϫ26 of the 1.63 MVSG promoter is essential. In terms of promoter activation, we believe that similarities to box 2 or box 1 might not be relevant.…”

Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

“…In addition, another distinct factor binds to an upstream promoter element and activates ribosomal DNA (rDNA) transcription by stabilizing PIC formation. The VSG ES promoter extends only 67 bp upstream of the transcription initiation site and consists of two short sequence elements (24,34). Since both elements are required for efficient binding of purified CITFA (1) and since they are so close to the initiation site, CITFA appears to be the functional equivalent of the yeast core factor and human SL1.…”