title: Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli.
abstract: RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.
authors.affiliation: Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, United States.
authors.forename: M Patrick
authors.initials: MP
authors.lastname: Hensley
authors.forename: Thusitha S
authors.initials: TS
authors.lastname: Gunasekera
authors.forename: J Allen
authors.initials: JA
authors.lastname: Easton
authors.forename: Tara K
authors.initials: TK
authors.lastname: Sigdel
authors.forename: Stacy A
authors.initials: SA
authors.lastname: Sugarbaker
authors.forename: Lindsey
authors.initials: L
authors.lastname: Klingbeil
authors.forename: Robert M
authors.initials: RM
authors.lastname: Breece
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D056928:X-Ray Absorption Spectroscopy / Q000379:methods; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D012269:Ribosomal Proteins; C575805:YkgM protein, E coli; C470844:ZinT protein, E coli; C488663:ZnuA protein, E coli; C050985:ribosomal protein L31; D006639:Histidine; D015032:Zinc; D003545:Cysteine