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J. Biol. Chem., Vol. 283, Issue 5, 2495-2507, February 1, 2008

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Control and Regulation of Gene Expression

QUANTITATIVE ANALYSIS OF THE EXPRESSION OF PHOSPHOGLYCERATE KINASE IN BLOODSTREAM FORM TRYPANOSOMA BRUCEI^*

Jurgen R. Haanstra¹, Mhairi Stewart, Van-Duc Luu, Arjen van Tuijl, Hans V. Westerhoff^¶, Christine Clayton², and Barbara M. Bakker²³

From the Vrije Universiteit, Biocentrum Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands, the Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany, and the ^¶Manchester Centre for Integrative Systems Biology, MIB, SCEAS, the University of Manchester, Manchester M1 7ND, United Kingdom

Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e.g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation.

Received for publication, July 13, 2007 , and in revised form, October 31, 2007.

^* This work was supported in part by a Vernieuwingsimpuls (to B. M. B.) from Netherlands Science Organization NWO, by the EC-FP6 program (through BioSim, NucSys, and EC-MOAN), the ESF program funcdyn, NWO-ALW and NWO-CLS, and by the Biotechnology and Biological Sciences Research Council (to H. V. W.). Work in the Clayton laboratory was supported by the Wellcome Trust (to M. S.) and the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 3B, discussion of Table 5, and additional references.

¹ Recipient of a VU-FALW Rijnland fonds travel grant for a visit of six months to the Zentrum für Molekulare Biologie der Universität Heidelberg.

² Both authors contributed equally to this manuscript.

³ To whom correspondence should be addressed. Tel.: 31-20-598-7196; Fax: 31-20-598-7229; E-mail: barbara.bakker{at}falw.vu.nl.

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