Emerging Functions of Transcription Factors in Malaria Parasite

Transcription is a process by which the genetic information stored in DNA is converted into mRNA by enzymes known as RNA polymerase. Bacteria use only one RNA polymerase to transcribe all of its genes while eukaryotes contain three RNA polymerases to transcribe the variety of eukaryotic genes. RNA polymerase also requires other factors/proteins to produce the transcript. These factors generally termed as transcription factors (TFs) are either associated directly with RNA polymerase or add in building the actual transcription apparatus. TFs are the most common tools that our cells use to control gene expression. Plasmodium falciparum is responsible for causing the most lethal form of malaria in humans. It shows most of its characteristics common to eukaryotic transcription but it is assumed that mechanisms of transcriptional control in P. falciparum somehow differ from those of other eukaryotes. In this article we describe the studies on the main TFs such as myb protein, high mobility group protein and ApiA2 family proteins from malaria parasite. These studies show that these TFs are slowly emerging to have defined roles in the regulation of gene expression in the parasite

Genetically engineered synthetic miniaturized versions of Plasmodium falciparum UvrD helicase are catalytically active.

Helicases catalyze unwinding of double stranded nucleic acids in an energy-dependent manner. We have reported characterization of UvrD helicase from Plasmodium falciparum. We reported that the N-terminal and C-terminal fragments of PfUvrD contain characteristic ATPase and DNA helicase activities. Here we report the generation and characterization of a genetically engineered version of PfUvrD and its derivatives. This synthetic UvrD (sUD) contains all the conserved domains of PfUvrD but only the intervening linker sequences are shortened. sUD (∼ 45 kDa) and one of its smallest derivative sUDN1N2 (∼ 22 kDa) contain ATPase and DNA helicase activities. sUD and sUDN1N2 can utilize hydrolysis of all the NTPs and dNTPs, can also unwind blunt end duplex DNA substrate and unwind DNA duplex in 3 to 5 direction only. Some of the properties of sUD are similar to the PfUvrD helicase. Mutagenesis in the conserved motif Ia indicate that the mutants sUDM and sUDN1N2M lose all the enzyme activities, which further confirms that these activities are intrinsic to the synthesized proteins. These studies show that for helicase activity only the conserved domains are essentially required and intervening sequences have almost no role. These observations will aid in understanding the unwinding mechanism by a helicase

Unwinding activity analysis of sUD and sUDN1N2 with different substrates.

<p>The structure of the substrate used is shown. Asterisk (*) denotes the ³²P-labeled end. A. Lanes 1–6 are the reactions with increasing concentration of sUD. B. Lanes 1–6 are the reactions with increasing concentration of sUDN1N2. C. Lanes 1–6 are the reactions with increasing concentration of sUD. D. Lanes 1–6 are the reactions with increasing concentration of sUDN1N2. In panel A–D, lane C is reaction without enzyme and lane B is heat treated substrate.</p

Figure 4

<p>A–D. Secondary structure representation. The protein sequences of sUD, sUDM, sUDN1N2 and sUDN1N2M were submitted to the server at <a href="http://bioinf.cs.ucl.ac.uk/psipred/and" target="_blank">http://bioinf.cs.ucl.ac.uk/psipred/and</a> the secondary structures were determined. The graphs in A–D represent the structures of sUD, sUDM, sUDN1N2 and sUDN1N2M, respectively. E. Circular dichroism (CD) spectra of sUD, sUDM, sUDN1N2 and sUDN1N2M. F-G. CD spectra of sUD and sUDM, and sUDN1N2 and sUDN1N2M, respectively at different temperature.</p

Structure modelling of synthetic UvrD.

<p>A–C. sUD sequence was submitted to Swissmodel server and the structure was obtained. A. Template B. sUD C. superimposed image. D. Secondary structure of sUD. E–G. sUDN1N2 sequence was submitted to Swissmodel server and the structure was obtained. E. Template; F. sUDN1N2; G. superimposed image. D. Secondary structure of sUDN1N2.</p

SDS PAGE and Western blot analysis.

<p>A and B. Coomasie blue stained gel. Lane M in A and B are protein molecular weight markers and the proteins loaded in each lane are written on top of the gels. C and D. Western blot analysis. Lane M in C and D are protein molecular weight markers and the proteins loaded in each lane are written on top of the gel.</p

Determination of nucleotide-dependence of helicase activity.

<p>A–B. Nucleotide requirement of helicase activity of sUD (A) and sUDN1N2 (B). Helicase activity of sUD and sUDN1N2 in the presence of NTP/dNTPs written on top of the gel. Lane C is enzyme reaction in the absence of any NTP or dNTP and B is heat denatured substrates. C–D. Helicase activity of sUD (C) and sUDN1N2 (D) using varying concentration of ATP written on top of the gel. Lane C is enzyme reaction without any ATP and B is heat denatured substrate. In panel A–D, the quantitative enzyme activity data from the autoradiogram are shown.</p

Analysis of ATPase and helicase activities after immunodepletion.

<p>A. ATPase activity of sUD. Lanes 1–3, reactions with increasing concentration of sUD pretreated with immune IgG, lanes 4–8 reactions with increasing concentration of sUD pretreated with pre-immune IgG. B. ATPase activity of sUDN1N2. Lanes 1–3, reactions with increasing concentration of sUDN1N2 pretreated with immune IgG, lanes 4–8 reactions with increasing concentration of sUDN1N2 pretreated with pre-immune IgG. Lane C in A and B is reaction without protein. C. Helicase activity of sUD. Lanes 1–4, reactions with increasing concentration of sUD pretreated with pre-immune IgG, lanes 5–8, reactions with increasing concentration of sUD pretreated with immune IgG. D. Helicase activity of sUDN1N2. Lanes 1–4, reactions with increasing concentration of sUDN1N2 pretreated with pre-immune IgG, lanes 5–8 reactions with increasing concentration of sUDN1N2 pretreated with immune IgG. In panel C and D, lane C is reaction without protein and lane B is heat treated substrate. In each panel the quantitative data from the autoradiogram are also shown.</p

Analysis of ATPase activity.

<p>A. Lane 1, sUD, lanes 2–4, increasing concentration of sUDN1, lanes 5–7, increasing concentration of sUDN2, lanes 8–10, increasing concentration of sUDC1, lanes 11–13, increasing concentration of sUDC2, and lanes 14–16, increasing concentration of sUDN2C1. B. Lane 1 contains sUD, lanes 2–3, increasing concentration of sUDC1C2, lanes 4–5, increasing concentration of sUDN1N2C1, lanes 6–7, increasing concentration of sUDN1N2C2. C. Concentration dependent ATPase activity of sUD, Lanes 1–8 are increasing concentration of sUD and concentration are labelled at top of the autoradiogram. D. Time dependence of ATPase activity of sUD. The time of incubation in minutes is mentioned at the top of the autoradiogram and C is the control reaction without enzyme. E. Concentration dependent ATPase activity of sUDN1N2, Lanes 1–7 are increasing concentration of sUDN1N2 and concentration are labelled at top of the autoradiogram. F. Time dependence of ATPase activity of sUDN1N2. The time of incubation in minutes is mentioned at the top of the autoradiogram. C in panels A–F is the control reaction without enzyme. see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090951#pone.0090951.s004" target="_blank">Figure S4</a>.</p