• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of rnaThe RNA SocietyeTOC AlertsSubscriptionsJournal HomeCSHL PressRNA
RNA. Nov 2000; 6(11): 1649–1659.
PMCID: PMC1370033

Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7.

Abstract

Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain.

Full Text

The Full Text of this article is available as a PDF (2.8M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alexander RW, Muralikrishna P, Cooperman BS. Ribosomal components neighboring the conserved 518-533 loop of 16S rRNA in 30S subunits. Biochemistry. 1994 Oct 11;33(40):12109–12118. [PubMed]
  • Bischof O, Kruft V, Wittmann-Liebold B. Analysis of the puromycin binding site in the 70 S ribosome of Escherichia coli at the peptide level. J Biol Chem. 1994 Jul 15;269(28):18315–18319. [PubMed]
  • Brimacombe R. RNA-protein interactions in the Escherichia coli ribosome. Biochimie. 1991 Jul-Aug;73(7-8):927–936. [PubMed]
  • Buck MA, Cooperman BS. Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes. Biochemistry. 1990 Jun 5;29(22):5374–5379. [PubMed]
  • Capel MS, Engelman DM, Freeborn BR, Kjeldgaard M, Langer JA, Ramakrishnan V, Schindler DG, Schneider DK, Schoenborn BP, Sillers IY, et al. A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli. Science. 1987 Dec 4;238(4832):1403–1406. [PubMed]
  • Cate JH, Yusupov MM, Yusupova GZ, Earnest TN, Noller HF. X-ray crystal structures of 70S ribosome functional complexes. Science. 1999 Sep 24;285(5436):2095–2104. [PubMed]
  • Clemons WM, Jr, May JL, Wimberly BT, McCutcheon JP, Capel MS, Ramakrishnan V. Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature. 1999 Aug 26;400(6747):833–840. [PubMed]
  • Dijk J, Littlechild JA, Freund AM, Pouyet J, Daune M, Provencher SW. The secondary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by circular dichroic spectroscopy. Biochim Biophys Acta. 1986 Nov 21;874(2):227–234. [PubMed]
  • Dontsova O, Dokudovskaya S, Kopylov A, Bogdanov A, Rinke-Appel J, Jünke N, Brimacombe R. Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues. EMBO J. 1992 Aug;11(8):3105–3116. [PMC free article] [PubMed]
  • Döring T, Mitchell P, Osswald M, Bochkariov D, Brimacombe R. The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop. EMBO J. 1994 Jun 1;13(11):2677–2685. [PMC free article] [PubMed]
  • Dragon F, Brakier-Gingras L. Interaction of Escherichia coli ribosomal protein S7 with 16S rRNA. Nucleic Acids Res. 1993 Mar 11;21(5):1199–1203. [PMC free article] [PubMed]
  • Dragon F, Payant C, Brakier-Gingras L. Mutational and structural analysis of the RNA binding site for Escherichia coli ribosomal protein S7. J Mol Biol. 1994 Nov 18;244(1):74–85. [PubMed]
  • Draper DE. Themes in RNA-protein recognition. J Mol Biol. 1999 Oct 22;293(2):255–270. [PubMed]
  • Ehresmann B, Reinbolt J, Backendorf C, Tritsch D, Ebel J. Studies of the binding sites of Escherichia coli ribosomal protein S7 with 16S RNA by ultraviolet irradiation. FEBS Lett. 1976 Sep 1;67(3):316–319. [PubMed]
  • Fredrick K, Dunny GM, Noller HF. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits. J Mol Biol. 2000 May 5;298(3):379–394. [PubMed]
  • Green R, Noller HF. Ribosomes and translation. Annu Rev Biochem. 1997;66:679–716. [PubMed]
  • Gregory RJ, Cahill PB, Thurlow DL, Zimmermann RA. Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA. J Mol Biol. 1988 Nov 20;204(2):295–307. [PubMed]
  • Gutell RR, Larsen N, Woese CR. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol Rev. 1994 Mar;58(1):10–26. [PMC free article] [PubMed]
  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. [PubMed]
  • Hosaka H, Nakagawa A, Tanaka I, Harada N, Sano K, Kimura M, Yao M, Wakatsuki S. Ribosomal protein S7: a new RNA-binding motif with structural similarities to a DNA architectural factor. Structure. 1997 Sep 15;5(9):1199–1208. [PubMed]
  • Huenges M, Rölz C, Gschwind R, Peteranderl R, Berglechner F, Richter G, Bacher A, Kessler H, Gemmecker G. Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein. EMBO J. 1998 Jul 15;17(14):4092–4100. [PMC free article] [PubMed]
  • Johanson U, Hughes D. Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli. Gene. 1992 Oct 12;120(1):93–98. [PubMed]
  • Kao-Huang Y, Revzin A, Butler AP, O'Conner P, Noble DW, von Hippel PH. Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: measurement of DNA-bound Escherichia coli lac repressor in vivo. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4228–4232. [PMC free article] [PubMed]
  • Karginov AV, Karginova OA, Spiridonova VA, Kopylov AM. In vivo assembly of plasmid-expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression. FEBS Lett. 1995 Aug 7;369(2-3):158–160. [PubMed]
  • Kuwano Y, Olvera J, Wool IG. The primary structure of rat ribosomal protein S5. A ribosomal protein present in the rat genome in a single copy. J Biol Chem. 1992 Dec 15;267(35):25304–25308. [PubMed]
  • Legault P, Li J, Mogridge J, Kay LE, Greenblatt J. NMR structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif. Cell. 1998 Apr 17;93(2):289–299. [PubMed]
  • Miyamoto A, Usui M, Yamasaki N, Yamada N, Kuwano E, Tanaka I, Kimura M. Role of the N-terminal region of ribosomal protein S7 in its interaction with 16S rRNA which binds to the concavity formed by the beta-ribbon arm and the alpha-helix. Eur J Biochem. 1999 Dec;266(2):591–598. [PubMed]
  • Mueller F, Brimacombe R. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. II. The RNA-protein interaction data. J Mol Biol. 1997 Aug 29;271(4):545–565. [PubMed]
  • Muralikrishna P, Cooperman BS. A photolabile oligodeoxyribonucleotide probe of the decoding site in the small subunit of the Escherichia coli ribosome: identification of neighboring ribosomal components. Biochemistry. 1994 Feb 15;33(6):1392–1398. [PubMed]
  • Nowotny V, Nierhaus KH. Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7. Biochemistry. 1988 Sep 6;27(18):7051–7055. [PubMed]
  • Powers T, Noller HF. Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA. RNA. 1995 Apr;1(2):194–209. [PMC free article] [PubMed]
  • Powers T, Changchien LM, Craven GR, Noller HF. Probing the assembly of the 3' major domain of 16 S ribosomal RNA. Quaternary interactions involving ribosomal proteins S7, S9 and S19. J Mol Biol. 1988 Mar 20;200(2):309–319. [PubMed]
  • Reinbolt J, Tritsch D. The primary structure of ribosomal protein S7 from E. coli strains K and B. FEBS Lett. 1978 Jul 15;91(2):297–301. [PubMed]
  • Rosen KV, Zimmerman RA. Photoaffinity labeling of 30S-subunit proteins S7 and S11 by 4-thiouridine-substituted tRNA(Phe) situated at the P site of Escherichia coli ribosomes. RNA. 1997 Sep;3(9):1028–1036. [PMC free article] [PubMed]
  • Saito K, Mattheakis LC, Nomura M. Post-transcriptional regulation of the str operon in Escherichia coli. Ribosomal protein S7 inhibits coupled translation of S7 but not its independent translation. J Mol Biol. 1994 Jan 7;235(1):111–124. [PubMed]
  • Saito K, Nomura M. Post-transcriptional regulation of the str operon in Escherichia coli. Structural and mutational analysis of the target site for translational repressor S7. J Mol Biol. 1994 Jan 7;235(1):125–139. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Spiridonova VA, Golovin AV, Drygin DYu, Kopylov AM. An extremely high conservation of RNA-protein S7 interactions during prokaryotic ribosomal biogenesis. Biochem Mol Biol Int. 1998 May;44(6):1141–1146. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Sylvers LA, Kopylov AM, Wower J, Hixson SS, Zimmermann RA. Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites. Biochimie. 1992 Apr;74(4):381–389. [PubMed]
  • Tan R, Frankel AD. Structural variety of arginine-rich RNA-binding peptides. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5282–5286. [PMC free article] [PubMed]
  • Tanaka I, Nakagawa A, Hosaka H, Wakatsuki S, Mueller F, Brimacombe R. Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA. RNA. 1998 May;4(5):542–550. [PMC free article] [PubMed]
  • Tocilj A, Schlünzen F, Janell D, Glühmann M, Hansen HA, Harms J, Bashan A, Bartels H, Agmon I, Franceschi F, et al. The small ribosomal subunit from Thermus thermophilus at 4.5 A resolution: pattern fittings and the identification of a functional site. Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14252–14257. [PMC free article] [PubMed]
  • Urlaub H, Kruft V, Bischof O, Müller EC, Wittmann-Liebold B. Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies. EMBO J. 1995 Sep 15;14(18):4578–4588. [PMC free article] [PubMed]
  • Urlaub H, Thiede B, Müller EC, Brimacombe R, Wittmann-Liebold B. Identification and sequence analysis of contact sites between ribosomal proteins and rRNA in Escherichia coli 30 S subunits by a new approach using matrix-assisted laser desorption/ionization-mass spectrometry combined with N-terminal microsequencing. J Biol Chem. 1997 Jun 6;272(23):14547–14555. [PubMed]
  • Vladimirov SN, Ivanov AV, Karpova GG, Musolyamov AK, Egorov TA, Thiede B, Wittmann-Liebold B, Otto A. Characterization of the human small-ribosomal-subunit proteins by N-terminal and internal sequencing, and mass spectrometry. Eur J Biochem. 1996 Jul 1;239(1):144–149. [PubMed]
  • Wimberly BT, White SW, Ramakrishnan V. The structure of ribosomal protein S7 at 1.9 A resolution reveals a beta-hairpin motif that binds double-stranded nucleic acids. Structure. 1997 Sep 15;5(9):1187–1198. [PubMed]
  • Zawadzki V, Gross HJ. Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 1991 Apr 25;19(8):1948–1948. [PMC free article] [PubMed]

Articles from RNA are provided here courtesy of The RNA Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...