Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

We have defined a core promoter element downstream of the TATA box that is recognized by TFIIB. This involves a DNA-binding domain in TFIIB that is distinct from the helix-turn-helix motif (which recognizes an element upstream of the TATA box). The TFIIB recognition element we describe regulates transcription in a manner that is promoter context-dependent, particularly with respect to the TFIIB recognition element that is located upstream of the TATA box. Thus TFIIB can recognize two distinct sequence elements that flank the TATA box, employing independent DNA-binding motifs and cooperating in the regulation of transcription. The core promoter of genes transcribed by RNA polymerase II (pol II) contains DNA sequence elements that are recognized by the general transcription machinery (Butler and Kadonaga 2002;Smale and Kadonaga 2003;Hahn 2004). These elements help to direct and orient the preinitiation complex (PIC) at the promoter. In addition, they play a critical role in the regulation of transcription (Hochheimer and Tjian 2003;Basehoar et al. 2004;Müller and Tora 2004). The best-characterized core promoter element is the TATA box, which is recognized by the TATA-binding protein (TBP) subunit of TFIID and nucleates PIC formation. However, many promoters do not contain a TATA box, and thus alternative mechanisms are employed to initiate PIC formation. The downstream promoter element (DPE) plays a major role at TATA-less promoters and is recognized by components of TFIID other than TBP. In addition, the initiator element (Inr), which spans the region where mRNA synthesis begins, can nucleate PIC formation via components of TFIID. The Inr can function independently or in combination with a TATA element, exerting a positive effect on transcription. In addition, a recently reported core promoter sequence, the motif-ten-element (MTE), can cooperate with the initiator to stimulate transcription (Lim et al. 2004).The general transcription factor TFIIB can also engage in sequence-specific contact with the core promoter (Lagrange et al. 1998;Qureshi and Jackson 1998). This TFIIB-recognition element (BRE) is located immediately upstream of the TATA element in a subset of promoters. A helix-turn-helix (HTH) motif at the C terminus of TFIIB mediates contact with the BRE (Lagrange et al. 1998;Tsai and Sigler 2000). The TFIIB-BRE interaction facilitates the assembly of a TFIIB-TBP-TATA complex (Lagrange et al. 1998;Qureshi and Jackson 1998). In addition, the TFIIB-BRE interaction can orient the PIC on the promoter (Bell et al. 1999;Littlefield et al. 1999;Tsai and Sigler 2000). The BRE acts to increase basal transcription in a system composed of purified factors (Lagrange et al. 1998). In crude nuclear extracts and in living cells, the BRE acts to suppress basal transcription, but has no effect on the level of transcription attained in the presence of an activator protein (Evans et al. 2001;Chen and Manley 2003). Interestingly, the HTH motif is not conserved in yeast or plant TFIIB (Lagrange et al. 1998;Tsai and Sigler 2000)...

Accurate transcription of a gene by RNA polymerase II requires the assembly of a group of general transcription factors at the promoter. The general transcription factor TFIIB plays a central role in preinitiation complex assembly, providing a bridge between promoter-bound TFIID and RNA polymerase II. TFIIB makes extensive contact with the core promoter via two independent DNA-recognition modules. In addition to interacting with other general transcription factors, TFIIB directly modulates the catalytic center of RNA polymerase II in the transcription complex. Moreover, TFIIB has been proposed as a target of transcriptional activator proteins that act to stimulate preinitiation complex assembly. In this review, we will discuss our current understanding of these activities of TFIIB.

SummarySex is an obligate step in the life cycle of the malaria parasite and occurs in the midgut of the mosquito vector. With both Plasmodium falciparum and Plasmodium berghei, the tryptophan metabolite xanthurenic acid induces the release of motile male gametes from red blood cells (exflagellation), a prerequisite for fertilization. The addition of cGMP or phosphodiesterase inhibitors to cultures of mature gametocytes has also been shown to stimulate exflagellation. Here, we demonstrate that there is a guanylyl cyclase activity associated with mature P. falciparum gametocyte membrane preparations, which is dependent on the presence of Mg 21 /Mn 21 but is inhibited by Ca 21 .