Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general pol II transcription factors have been elusive. The protozoan parasite Trypanosoma brucei branched early in eukaryotic evolution and is probably the most divergent well-studied eukaryote. Many discoveries of general interest have been made in T. brucei, emphasizing its value for understanding the evolution of core molecular processes.Transcription of protein-coding genes by RNA polymerase II (pol II) in T. brucei differs in two important aspects from most other eukaryotes. First, transcription is polycistronic: Arrays of sometimes >100 genes are transcribed in polycistronic transcription units (PTUs). This organization is reminiscent of operons in prokaryotes except that there is no evidence to suggest clustering of functionally related genes in T. brucei. Second, mRNAs are separated post-transcriptionally by coupled splicing and polyadenylation reactions that add a 39-nucleotide (nt) ''spliced-leader'' to every mRNA (for review, see Liang et al. 2003). Within a PTU, genes are transcribed from the same strand, but transcription of two neighboring PTUs can either be convergent or divergent. The regions between PTUs are referred to as strand switch regions (SSRs). Strand-specific nuclear run-on assays performed in Leishmania (Martinez-Calvillo et al. 2003), a genus related to T. brucei, have shown that pol II transcription starts at SSRs between two transcriptionally divergent PTUs (divergent SSRs) and ends at SSRs between two transcriptionally convergent PTUs (convergent SSRs). Because 75% of all Leishmania major genes can be found in the same genomic context in T. brucei (El-Sayed et al. 2005), indicating a high degree of synteny, it is reasonable to hypothesize that divergent SSRs in T. brucei are transcription start s...

Evolvability—the capacity to generate beneficial heritable variation—is a central property of biological systems. However, its origins and modulation by environmental factors have not been examined systematically. Here, we analyze the fitness effects of all single mutations in TEM-1 β-lactamase (4,997 variants) under selection for the wild-type function (ampicillin resistance) and for a new function (cefotaxime resistance). Tolerance to mutation in this enzyme is bimodal and dependent on the strength of purifying selection in vivo, a result that derives from a steep non-linear ampicillin-dependent relationship between biochemical activity and fitness. Interestingly, cefotaxime resistance emerges from mutations that are neutral at low levels of ampicillin but deleterious at high levels; thus the capacity to evolve new function also depends on the strength of selection. The key property controlling evolvability is an excess of enzymatic activity relative to the strength of selection, suggesting that fluctuating environments might select for high-activity enzymes.

Transcription of protein-coding genes in trypanosomes is polycistronic and gene expression is primarily regulated by post-transcriptional mechanisms. Sequence motifs in the untranslated regions regulate mRNA trans-splicing and RNA stability, yet where UTRs begin and end is known for very few genes. We used high-throughput RNA-sequencing to determine the genome-wide steady-state mRNA levels (‘transcriptomes’) for ∼90% of the genome in two stages of the Trypanosoma brucei life cycle cultured in vitro. Almost 6% of genes were differentially expressed between the two life-cycle stages. We identified 5′ splice-acceptor sites (SAS) and polyadenylation sites (PAS) for 6959 and 5948 genes, respectively. Most genes have between one and three alternative SAS, but PAS are more dispersed. For 488 genes, SAS were identified downstream of the originally assigned initiator ATG, so a subsequent in-frame ATG presumably designates the start of the true coding sequence. In some cases, alternative SAS would give rise to mRNAs encoding proteins with different N-terminal sequences. We could identify the introns in two genes known to contain them, but found no additional genes with introns. Our study demonstrates the usefulness of the RNA-seq technology to study the transcriptional landscape of an organism whose genome has not been fully annotated.

Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence-encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine.