Structure of the C-terminal domain of transcription factor IIB from Trypanosoma brucei.

In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB (tTFIIB(C)). The tTFIIB(C) structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIB(C) contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: substrate specificity studies on the recombinant and endogenous proteins.

A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5'-mRNA cap, i.e., m(7)GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m(7)GMP (or m(2,7)GMP) as one of the reaction products. Interestingly, m(7)Gpppm(3)(N6, N6, 2'O)A was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m(7)Gpppm(3)(N6, N6, 2'O)Apm(2'O)Apm(2'O)Cpm(2)(N3, 2'O)U, that occurs in these organisms.

Trypanosomal TBP functions with the multisubunit transcription factor tSNAP to direct spliced-leader RNA gene expression.

Protein-coding genes of trypanosomes are mainly transcribed polycistronically and cleaved into functional mRNAs in a process that requires trans splicing of a capped 39-nucleotide RNA derived from a short transcript, the spliced-leader (SL) RNA. SL RNA genes are individually transcribed from the only identified trypanosome RNA polymerase II promoter. We have purified and characterized a sequence-specific SL RNA promoter-binding complex, tSNAP(c), from the pathogenic parasite Trypanosoma brucei, which induces robust transcriptional activity within the SL RNA gene. Two tSNAP(c) subunits resemble essential components of the metazoan transcription factor SNAP(c), which directs small nuclear RNA transcription. A third subunit is unrelated to any eukaryotic protein and identifies tSNAP(c) as a unique trypanosomal transcription factor. Intriguingly, the unusual trypanosome TATA-binding protein (TBP) tightly associates with tSNAPc and is essential for SL RNA gene transcription. These findings provide the first view of the architecture of a transcriptional complex that assembles at an RNA polymerase II-dependent gene promoter in a highly divergent eukaryote.

RNA interference as a genetic tool in trypanosomes.

RNA interference (RNAi) is a cellular mechanism that is often exploited as a technique for quelling the expression of a specific gene. RNAi studies are carried out in vivo, making this a powerful means for the study of protein function in situ Several trypanosomatids, including those organisms responsible for human and animal diseases, naturally possess the machinery necessary for RNAi manipulations. This allows for the use of RNAi in unraveling many of the pressing questions regarding the parasite's unique biology. The completion of the Trypanosoma brucei genome sequence, coupled with several powerful genetic tools, has resulted in widespread utilization of RNAi in this organism. The key steps for RNAi-based reduction of gene expression, including parasite cell culture, DNA transfection, RNAi expression, and experimental execution, are discussed with a focus on procyclic forms of Trypanosoma brucei.

Gene transcription in trypanosomes.

Trypanosoma brucei and the other members of the trypanosomatid family of parasitic protozoa, contain an unusual RNA polymerase II enzyme, uncoordinated mRNA 5' capping and transcription initiation events, and most likely contain an abridged set of transcription factors. Pre-mRNA start sites remain elusive. In addition, two important life cycle stage-specific mRNAs are transcribed by RNA polymerase I. This review interprets these unusual transcription traits in the context of parasite biology.

Regulation of surface coat exchange by differentiating African trypanosomes.

African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

The characterization of mutant Bacillus subtilis adenylosuccinate lyases corresponding to severe human adenylosuccinate lyase deficiencies.

Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR). Several point mutations in the gene encoding the enzyme have been implicated in human disease. Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency. Site-directed mutagenesis was utilized to construct amino acid substitutions in B. subtilis adenylosuccinate lyase; Met(10), Ile(123), and Thr(367) were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli. These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters. The major deviations from the wild-type-like behavior were observed upon biophysical characterization. All of these enzymes with amino acid replacements are associated with marked thermal instability. I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation. T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis. Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired. The enzyme instability was found to be lessened by subunit complementation with the wild-type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients. Residues Met(10), Ile(123), and Thr(367) appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme.

The evolution of metabolic enzymes in Plasmodium and trypanosomatids as compared to Saccharomyces and Schizosaccharomyces.

Understanding how the biological connectivity of genes and gene products affects evolution is an important aspect of understanding evolution. Genes encoding enzymes are frequently used to carry out such analyses. Interestingly, studies have shown that connectivity in the metabolic networks in parasitic protists, including Plasmodium falciparum and Trypanosoma brucei, have been substantially altered as compared to free living eukaryotes, such as Saccharomyces cerevisiae. Herein, we have determined K(a) values, which are a measure of the non-synonymous substitution rate, and used them to examine the differences between the evolution of genes in T. brucei, P. falciparum, S. cerevisiae, and Schizosaccharomyces pombe. All four organisms share similar traits with respect to the evolution of genes encoding metabolic enzymes. First, genes encoding metabolic enzymes have lower K(a) values than genes encoding non-metabolic proteins. In addition, perturbations of the metabolic network appear to have limited affects on the genes encoding enzymes near the perturbation. In most cases, there is a negative relationship between connectivity in the metabolic network of the gene product and the K(a) value for the gene, i.e. examining how much constraint there is on gene evolution when it is connected to many other genes. In addition, we find that the K(a) values of orthologs encoding for metabolic enzymes in each organism are significantly correlated, indicating similar patterns of non-synonymous substitutions. In total, our results indicate that the evolution of genes encoding metabolic enzymes do not tend to be greatly affected by changes in the metabolic network.

TbENF is an essential TbTFIIB-interacting trypanosomatid-specific factor.

Trypanosoma brucei, the causative agent of African Sleeping Sickness, is replete with unique biochemistry, including unusual features of gene transcription. The parasite also contains over 4500 non-annotated genes, representing novel biochemistry yet to be explored. Using tandem affinity purification (TAP)-tagged TbTFIIB, we identified and subsequently confirmed, one of the non-annotated T. brucei proteins, Tb11.02.4300, as a TbTFIIB-interacting protein. The 49 kDa protein is nuclear and essential for parasite variability as determined by RNA interference studies; hence, the nomenclature T. brucei Essential Nuclear Factor (TbENF). TbENF is shown to interact with DNA in a sequence-independent fashion under the conditions examined. Furthermore, TbENF bears motifs associated with many eukaryotic transcription factors, such as a glutamine-rich region and a leucine zipper, yet TbENF is specific to trypanosomatids making it a potentially attractive therapeutic target. Taken together, our results suggest a role for TbENF in trypanosome gene transcription.

Chromatin immunoprecipitation and microarray analysis reveal that TFIIB occupies the SL RNA gene promoter region in Trypanosoma brucei chromosomes.

RNA polymerase II (RNAP-II) synthesizes the m(7)G-capped Spliced Leader (SL) RNA and most protein-coding mRNAs in trypanosomes. RNAP-II recruitment to DNA usually requires a set of transcription factors that make sequence-specific contacts near transcriptional start sites within chromosomes. In trypanosomes, the transcription factor TFIIB is necessary for RNAP-II-dependent SL RNA transcription. However, the trypanosomal TFIIB (tTFIIB) lacks the highly basic DNA binding region normally found in the C-terminal region of TFIIB proteins. To assess the precise pattern of tTFIIB binding within the SL RNA gene locus, as well as within several other loci, we performed chromatin immunoprecipitation/microarray analysis using a tiled gene array with a probe spacing of 10 nucleotides. We found that tTFIIB binds non-randomly within the SL RNA gene locus mainly within a 220-nt long region that straddles the transcription start site. tTFIIB does not bind within the small subunit (SSU) rRNA locus, indicating that trypanosomal TFIIB is not a component of an RNAP-I transcriptional complex. Interestingly, discrete binding sites were observed within the putative promoter regions of two loci on different chromosomes. These data suggest that although trypanosomal TFIIB lacks a highly basic DNA binding region, it nevertheless localizes to discrete regions of chromatin that include the SL RNA gene promoter.

The characterization of a unique Trypanosoma brucei ß-hydroxybutyrate dehydrogenase.

A putative ß-hydroxybutyrate dehydrogenase (ßHBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble ßHBDH than to membrane-bound Type I ßHBDH found in higher eukaryotes. The ßHBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His(10)-TbßHBDH) was purified to approximate homogeneity from E. coli. ßHBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to D-3-hydroxybutyrate. In the direction of D-3-hydroxybutyrate formation, His(10)-TbßHBDH has a k(cat) value of 0.19 s(-1) and a K(M) value of 0.69 mM for acetoacetate. In the direction of acetoacetate formation, His(10)-TbßHBDH has a k(cat) value of 11.2 s(-1) and a K(M) value of 0.65 mM for D-3-hydroxybutyrate. Cofactor preference was examined and His(10)-TbßHBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized ßHBDHs. Furthermore, His(10)-TbßHBDH binds NAD(P)(+) in a cooperative fashion, another unique characteristic of trypanosome ßHBDH. The apparent native molecular weight of recombinant His(10)-TbßHBDH is 112 kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbßHBDH in vivo. Upon knockdown of TbßHBDH, a small reduction in parasite growth was observed suggesting ßHBDH has an important physiological role in T. brucei.

A divergent transcription factor TFIIB in trypanosomes is required for RNA polymerase II-dependent spliced leader RNA transcription and cell viability.

Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.

Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role.

Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.