The haem detoxification pathway of the malaria parasite Plasmodium falciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is polymerized by the parasite to form haemozoin pigment. Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2) has been implicated as the catalytic scaffold for detoxification of haem in the malaria parasite. Previously we have shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in Pfhrp-2, may be the major haem binding site in this protein. The haem binding studies carried out by ourselves indicate that up to 18 equivalents of haem could be bound by this protein with an observed K(d) of 0.94 microM. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to Pfhrp-2. This was supported by the K(d) value, of 37 nM, observed for the haem-chloroquine complex. The native PAGE studies reveal that the formation of the haem-Pfhrp-2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to Pfhrp-2. Moreover, the higher affinity of chloroquine for haem than Pfhrp-2 suggests a possible mechanism of action for chloroquine; it may remove the haem bound to Pfhrp-2 and form a complex that is toxic to the parasite.
Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.T ranscription of genes by RNA polymerase II (RNAPII) is a well-orchestrated process that involves steps of initiation, elongation, and termination. Following promoter clearance, RNAPII enters the phase of productive elongation that is achieved by a Brownian ratchet mechanism, in which the RNAPII oscillates between a pretranslocated and a posttranslocated state. After nucleotide addition to the 3= end of the RNA, the incoming nucleoside triphosphate (NTP) locks RNAPII in a posttranslocated form, readying it for the next cycle (1-4). However, productive elongation is not a product of efficient addition of nucleotides by RNAPII alone. During transcription elongation, RNAPII encounters several blocks, including sequence-specific pause sites, nucleotide limitations, DNA lesions, negative elongation factors, and DNA-bound proteins, which cause RNAPII to pause, arrest, or terminate transcription (5, 6). A myriad of elongation factors helps rescue paused/arrested polymerases and stimulate transcription (7,8). Each elongation factor acts via a different mechanism, and often, one works in combination with others.One of the best-characterized elongation factors known to rescue backtracked RNAPII is TFIIS. TFIIS promotes transcription elongation by stimulating the nucleolytic activity of RNAPII and realigning the 3= end of the transcript in the active site of arrested RNAPII (for reviews, see references 9 and 10). New evidence suggests that the cleavage-promoting activity of TFIIS is not the only way in which it stimulates elongation (11-13). Biochemical and biophysical studies have only recently begun to uncover the mechanisms by which TFIIS functions with TFII...
Background: mRNA transcription and decay are coordinated processes. Results: The Rpb4/7 module of RNA polymerase II is required for the transcription and mRNA decay factor Ccr4-Not to associate with elongation complexes. Conclusion: Association between these two entities is required for Ccr4-Not to promote transcription elongation. Significance: Our work provides molecular insights into how transcription and mRNA decay are linked.
RNA editing in the sleeping sickness parasite Trypanosoma brucei remodels mitochondrial transcripts by the addition and deletion of uridylates as specified by guide RNAs. Editing is catalyzed by at least three distinct ;20S multiprotein editosomes, all of which contain KREPB4, a protein with RNase III and Pumilio motifs. RNAi repression of KREPB4 expression in procyclic forms (PFs) strongly inhibited growth and in vivo RNA editing, greatly diminished the abundance of 20S editosomes, reduced cellular levels of editosome proteins, and generated ;5-10S editosome subcomplexes. Editing TUTase, exoUase, and RNA ligase activities were largely shifted from ;20S to ;5-10S fractions of cellular lysates. Insertion and deletion endonuclease activities in ;20S fractions were strongly reduced upon KREPB4 repression but were not detected in the 5-10S subcomplex fraction. Abundance of MRP1 and RBP16 proteins, which appear to be involved in RNA processing but are not components of the 20S editosome, was unaltered upon KREPB4 repression. These data suggest that KREPB4 is important for the structural integrity of ;20S editosomes, editing endonuclease activity, and the viability of PF T. brucei cells.
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