RESUMO
Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Leishmania major/enzimologia , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Cromatografia de Afinidade/métodos , RNA Polimerases Dirigidas por DNA/metabolismo , Genes de Protozoários/genética , Leishmania major/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Protozoário/genética , Transcrição GênicaRESUMO
The various Leishmania species are flagellated protozoans, responsible for a wide spectrum of human diseases. The sequence of the L. major genome is nearing completion and a large proportion of the identified genes have yet to be ascribed functions. DNA microarrays containing PCR-amplified DNA from a random amplified genomic library of L. major Friedlin (LmjF) [Mol. Biochem. Parasitol. 113 (2001) 337] were hybridized with fluorescent probes made from L. major Friedlin RNA from five time-points during differentiation from procyclics to metacyclics. The data were normalized for background and probe intensity and the relative abundance of RNA for each spot was calculated. Almost 15% (1387/9282) of the DNAs showed statistically significant (P<0.01) changes in expression (1.1-5-fold) during the transition, with 1.16% (108) showing up-regulation at two or more time-points and 0.14% (13) showing down-regulation. Northern blot analyses of selected genes confirmed these results. These studies confirmed the stage-specific expression of several known genes, as well as identifying a number of novel genes that are up-regulated in either procyclics or metacyclics.