Journal of Molecular Biology
Structural Basis of the Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA☆
Introduction
Proline is used as a source of carbon, nitrogen, and energy through two oxidative steps catalyzed by proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH).1, 2, 3, 4, 5, 6, 7 In enteric bacteria such as Escherichia coli, proline utilization requires two genes, putP and putA. The former encodes the PutP high-affinity Na +-proline transporter, and the latter encodes the multifunctional flavoprotein proline utilization A (PutA).8, 9 PutA is unique in that it functions both as a transcriptional repressor of the put genes and as a membrane-associated bifunctional proline catabolic enzyme.2, 10, 11, 12 The enzymatic and transport functions of the putA and putP genes, respectively, are conserved among different Gram-negative bacteria, whereas the genetic organization and regulatory mechanisms that control the expression of these genes are highly divergent.7, 10, 11, 12, 13, 14, 15, 16, 17, 18 The focus of this work is to provide a molecular and structural understanding of the regulation of put genes in E. coli by PutA.
PutA from E. coli combines PRODH, P5CDH, and transcriptional regulatory activities into a single polypeptide of 1320 amino acids.2, 19 Insights into the organization of the functional domains in PutA have been gained from molecular dissection and characterization of truncated PutA proteins. The PRODH and P5CDH active sites are located within residues 261–612 and 650–1130, respectively, with the PRODH active site utilizing a FAD cofactor and with P5CDH activity requiring NAD +. Structural studies have shown that the PRODH domain forms a unique (βα)8 barrel,20, 21 and that reduction by dithionite causes dramatic conformational changes in the FAD ribityl chain.22 Molecular dissection studies showed that the DNA-binding domain is contained in residues 1–47.23 Subsequently, the crystal structure of PutA52 (a polypeptide corresponding to residues 1–52 of E. coli PutA) was solved, showing that PutA is a member of the ribbon–helix–helix (RHH) family of transcriptional regulators.23, 24
While knowledge of PutA structure and function continues to build, a considerable gap in our understanding of critical PutA–DNA interactions in the put control DNA region remains. To further understand the regulation of proline metabolism in E. coli, we have identified the PutA binding sites in the put-regulatory region, elucidated the roles of these operators in repressing the expression of putA and putP, determined the crystal structure of PutA52 bound to one of the identified operators, and investigated the thermodynamics of DNA binding to PutA52 using isothermal titration calorimetry (ITC).
Section snippets
Identification of PutA binding sites
Initial localization of PutA binding sites in the put control DNA region was performed by gel mobility shift assays using different fragments of the 419-bp put control DNA. Systematic evaluation of different regions of the put control DNA indicated that PutA does not bind to the 1- to 170-bp region immediately downstream of putP (Fig. 1a, lanes 3 and 4). However, PutA was observed by gel mobility shift assays to bind to regions 183–231 and 342–412 of the put control DNA (data not shown).
Transcriptional regulation of the put regulon
Based on the arrangement of the five PutA–DNA binding sites, PutA most likely represses the put genes by hindering the σ70-dependent binding of E. coli RNA polymerase to the putA and putP promoter regions.32 We did not find additional PutA consensus binding sites in the coding regions of putA and putP, indicating that PutA binds only to the put intergenic region. Previous reports suggested that proline, via PutA, regulates the expression of putA more tightly than putP.25, 27 Here we have shown
Materials
Chemicals and buffers were purchased from Fisher Scientific and Sigma-Aldrich, Inc., unless otherwise stated. Restriction endonucleases and T4 DNA ligase were purchased from Fermentas and Invitrogen, respectively. BCA reagents used for protein quantitation were obtained from Pierce. Goat anti-rabbit secondary antibody was purchased from Amersham, Inc. E. coli strains XL-blue and BL21 DE3 pLysS were purchased from Stratagene. E. coli strain JT31 putA− lacZ− was a generous gift from J. Wood
Acknowledgements
This research was supported by National Institutes of Health grants GM065546 (J.J.T.) and GM061068 (D.F.B.), and National Science Foundation grant MCB0091664 (D.F.B.). C.A.B. was supported by a postdoctoral fellowship from the National Library of Medicine (2-T15-LM07089-14). We thank Damian Coventry and Paul Bourke for providing the computer code used in penetration depth calculations. Part of this research was performed at the Advanced Light Source, which is supported by the Director, Office
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The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
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Y.Z. and J.D.L. contributed equally to this research.