The Non-Canonical CTD of RNAP-II Is Essential for Productive RNA Synthesis in Trypanosoma brucei

The carboxy-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II (RNAP-II) is essential for gene expression in metazoa and yeast. The canonical CTD is characterized by heptapeptide repeats. Differential phosphorylation of canonical CTD orchestrates transcriptional and co-transcriptional maturation of mRNA and snRNA. Many organisms, including trypanosomes, lack a canonical CTD. In these organisms, the CTD is called a non-canonical CTD or pseudo-CTD (ΨCTD. In the African trypanosome, Trypanosoma brucei, the ΨCTD is ∼285 amino acids long, rich in serines and prolines, and phosphorylated. We report that T. brucei RNAP-II lacking the entire ΨCTD or containing only a 95-amino-acid-long ΨCTD failed to support cell viability. In contrast, RNAP-II with a 186-amino-acid-long ΨCTD maintained cellular growth. RNAP-II with ΨCTD truncations resulted in abortive initiation of transcription. These data establish that non-canonical CTDs play an important role in gene expression

Ab Initio Identification of Novel Regulatory Elements in the Genome of Trypanosoma brucei by Bayesian Inference on Sequence Segmentation

Background: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes. Methodology/Principle Findings: Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site. Conclusions/Significance: The nature of the elements discovered reinforces the hypothesis that context dependant RN

Polysomal mRNA Association and Gene Expression in <i>Trypanosoma brucei</i>

Background: The contrasting physiological environments of Trypanosoma brucei procyclic (insect vector) and bloodstream (mammalian host) forms necessitates deployment of different molecular processes and, therefore, changes in protein expression. Transcriptional regulation is unusual in T. brucei because the arrangement of genes is polycistronic; however, genes which are transcribed together are subsequently cleaved into separate mRNAs by trans-splicing. Following pre-mRNA processing, the regulation of mature mRNA stability is a tightly controlled cellular process. While many stage-specific transcripts have been identified, previous studies using RNA-seq suggest that changes in overall transcript level do not necessarily reflect the abundance of the corresponding protein. Methods: To better understand the regulation of gene expression in T. brucei, we performed a bioinformatic analysis of RNA-seq on total, sub-polysomal, and polysomal mRNA samples. We further cross-referenced our dataset with a previously published proteomics dataset to identify new protein coding sequences. Results: Our analyses showed that several long non-coding RNAs are more abundant in the sub-polysome samples, which possibly implicates them in regulating cellular differentiation in T. brucei. We also improved the annotation of the T.brucei genome by identifying new putative protein coding transcripts that were confirmed by mass spectrometry data. Conclusions: Several long non-coding RNAs are more abundant in the sub-polysome cellular fractions and might pay a role in the regulation of gene expression. We hope that these data will be of wide general interest, as well as being of specific value to researchers studying gene regulation expression and life stage transitions in T. brucei

Development of an ultrathin liquid sheet target for laser ion acceleration at high repetition rates in the kilohertz range

A colliding microjet liquid sheet target system was developed and tested for pairs of round nozzles of 10, 11 and 18 μm in diameter. The sheet’s position stability was found to be better than a few micrometers. Upon interaction with 50 mJ laser pulses, the 18 μm jet has a resonance amplitude of 16 μm at a repetition rate of 33 Hz, while towards 100 Hz it converges to 10 μm for all nozzles. A white-light interferometric system was developed to measure the liquid sheet thickness in the target chamber both in air and in vacuum, with a measurement range of 182 nm–1 μm and an accuracy of ±3%. The overall shape and 3D shape of the sheet follow the Hasson–Peck model in air. In vacuum versus air, the sheet gradually loses 10% of its thickness, so the thinnest sheet achieved was below 200 nm at a vacuum level of 10–4 mbar, and remained stable for several hours of operation

RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor

Spliced leader RNA transcription is essential for cell viability in trypanosomes. The SL RNA genes are expressed from the only defined RNA polymerase II-dependent promoter identified to date in the trypanosome genome. The SL RNA gene promoter has been shown by in vitro and in vivo analyses to have a tripartite architecture. The upstream most cis-acting element, called PBP-1E, is located between 70 and 60 bp upstream from the transcription start site. This essential element functions along with two downstream elements to direct efficient and proper initiation of transcription. Electrophoretic mobility-shift studies detected a 122-kDa protein, called PBP-1, which interacts with PBP-1E. This protein is the first sequence-specific, double-stranded DNA-binding protein isolated in trypanosomes. Three polypeptides copurify with PBP-1 activity, suggesting that PBP-1 is composed of 57-, 46-, and 36-kDa subunits. We have cloned the genes that encode the 57- and 46-kDa subunits. The 46-kDa protein is a previously uncharacterized protein and may be unique to trypanosomes. Its predicted tertiary structure suggests it binds DNA as part of a complex. The 57-kDa subunit is orthologous to the human small nuclear RNA-activating protein (SNAP)50, which is an essential subunit of the SNAP complex (SNAPc). In human cells, SNAPc binds to the proximal sequence element in both RNA polymerase II- and III-dependent small nuclear RNA gene promoters. These findings identify a surprising link in the transcriptional machinery across a large evolutionary distance in the regulation of small nuclear RNA genes in eukaryotes

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 Å resolution structure of the C-terminal domain of tTFIIB (tTFIIBC). The tTFIIBC 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, tTFIIBC 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