A high-expression system of L11 was constructed and investigated its interaction with other elements of the ribosome using physicochemical methods. The gene rplK, coding for the protein L11 from the E. coli 50S ribosomal subunit was amplifyied, cloned and over-expressed. The protein L11 was purified under native and denaturing conditions, refolded and the structure of both proteins was compared. The protein L11 properly refolded from 6M urea after dialysis. Experiments on binding of proteins L11, RRF and EF-G from Escherichia coli were performed by ana-lytical centrifugation and Biacore. Specific binding between protein L11 and RRF by analytical cen-trifugation was not detected probably due to struc-tural reasons. These findings may be helpful in the design of new antibiotics that specifically disrupt the interactions in the “GTP-associated site” of the bac-terial ribosome, as many of them are not effective anymore. A common intrinsically disordered region of protein L11 was found to be the amino acid se-quence 86-97, while the residues 67-74, containing the linker region, are predicted to be disordered by DisEMBL.
KeywordsCloning Of L11Rplk GenePurification Under Denaturing ConditionRefoldingInteraction With RibosomeThiostreptonIntrinsic Disorder
Choli, T. (1989) Structural properties of ribosomal protein L11 from Escherichia coli. Biochemistry International, 19, 1323-1338.
Demirci, H., Gregory, S.T., Dahlberg, A.E. and Jogl, G. (2007) Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA. The EMBO Journal, 26, 567-77. doi:10.1038/sj.emboj.7601508
Gudkov, A.T. and Tumanova, L.G. (1979) A physic-chemical characteristic of ribosomal proteins L10 and L11. Molecular Biology, 14, 798-802.
Tumanova, L.G., Gudkov, A.T., Bushuev, V.N. and Okon, M.S. (1981) Study of the tertiary structure of protein L11 from Escherichia coli ribosomes in solution by proton magnetic resonance. FEBS Letters, 127, 241-244. doi:10.1016/0014-5793(81)80215-3
Kime, M.J., Tatcliffe, R.G., Moore, P.B. and Williams, R.J.P. (1980) On the renaturation of ribosomal protein L11. European Journal of Biochemistry, 110, 493-498. doi:10.1111/j.1432-1033.1980.tb04891.x
Khechinashvili, N.N., Koteliansky, V.E., Gogia, Z.V., Littlechild, J. and Dijk, J. (1978) A heat denaturation study of several ribosomal proteins from Escherichia coli by scanning microcalorimetry. FEBS Letters, 95, 270-272. doi:10.1016/0014-5793(78)81008-4
McGuffin, L.J., Bryson, K. and Jones, D.T. (2000) The PSIPRED protein structure prediction server. Bioinformatics, 16, 404-405. doi:10.1093/bioinformatics/16.4.404
Hinck, A.P., Markus, M.A., Huang, S., Grzesiek, S., Kustonovich, I., Draper, D.E. and Torchia, D.A. (1997) The RNA binding domain of ribosomal protein L11: Three-dimensional structure of the RNA-bound form of the protein and its interaction with 23S rRNA. Journal of Molecular Biology, 274, 101-113. doi:10.1006/jmbi.1997.1379
Serdyuk, I.N. (2007) Structured proteins and proteins with intrinsic disorder. Molecular Biology, 41, 262-277.
Dunker, A.K., Brown, C.J., Lawson, J.D., Iakoucheva, L.M. and Obradovic, Z. (2002) Intrinsic disorder and protein function. Biochemistry, 41, 6573-6582. doi:10.1021/bi012159+
Uversky, V.N., Gillespie, J.R. and Fink, A.L. (2000) Why are “natively unfolded” proteins unstructured under physiologic conditions? Proteins, 41, 415-427. doi:10.1002/1097-0134(20001115)41:3 3.0.CO;2-7
Markus, M.A., Hinck, A.P., Huang, S., Draper, D.E. and Torchia, D.A. (1997) High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that becomes structured upon binding to RNA. Nature Structural Biology, 4, 70-77. doi:10.1038/nsb0197-70
Linding, R., Jensen, L.J., Diella, F., Bork, P., Gibson, T.J. and Russell, R.B. (2003) Protein disorder prediction: Implications for structural proteomics. Structure, 11, 1316-1317.
Dosztanyi, Z., Csizmok, V., Tompa, P. and Simon, I. (2005) IUPred: Web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics, 21, 3433-3434. doi:10.1093/bioinformatics/bti541
Prilusky, J., Felder, C.E., Zeev-Ben-Mordehai, T., Rydberg, E.H., Man, O., Beckmann, J.S., Silman, I. and Sussman, J.L. (2005) FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics, 21, 3435-3438. doi:10.1093/bioinformatics/bti537
Yang, Z.E., Thomson, R., McNeil, P. and Esnouf, R.M. (2005) RONN: The bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics, 21, 3369-3376. doi:10.1093/bioinformatics/bti534
Wimberly, B.T., Guymon, R., McCutcheon, J.P., White, S.W. and Ramacrishnan, V. (1999) A detailed view of a ribosomal active site: the structure of the L11-RNA complex. Cell, 97, 491-502. doi:10.1016/S0092-8674(00)80759-X
Jiangang, L., Narayanan, B.P., Christopher, J.O., Eric, W.S., Uversky, V.N. and Dunker, A.K. (2006) Intrinsic disorder in transcription factors. Biochemistry, 45, 6873-6888. doi:10.1021/bi0602718
Harms, M., Wilson, D.N., Schluenzen, F., Connell, S.R., Stachelhaus, T., Zaborowska, Z., Spahn, C.M. and Fucini, P. (2008) Translational regulation via L11: Molecular switches on the ribosome turned on and off by thiostrepton and micrococcin. Molecular Cell, 30, 26-38. doi:10.1016/j.molcel.2008.01.009
Lee, D., Walsh, J.D., Yu, P., Markus, M.A., Choli-Papadopoulou, T., Schwieters, C.D., Krueger, S., Draper, D.E. and Wang, Y.X. (2007) The structure of free L11 and functional dynamics of L11 in free, L11 rRNA(58 nt)binary and L11-rRNA(58 nt)-thiostrepton ternary complexes. Journal of Molecular Biology, 367, 1007-1022. doi:10.1016/j.jmb.2007.01.013
Jenvert, R.M. and Schiavone, L.H. (2007) The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor. Journal of Molecular Biology, 365, 764-772. doi:10.1016/j.jmb.2006.10.065
Iben, J.R. and Draper, D.E. (2008) Specific interactions of the L10.(L12)4 ribosomal protein complex with mRNA, rRNA,and L11. Biochemistry, 47, 2721-2731. doi:10.1021/bi701838y
Jonker, H.R., Ilin, S., Grimm, S.K., W?hnert, J. and Schwalbe, H. (2007) L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy. Nucleic Acids Research, 35, 441-454. doi:10.1093/nar/gkl1066
Ilin, S., Hoskins, A., Ohlenschl?ger, O., Jonker, H.R., Schwalbe, H. and W?hnert, J. (2005) Domain reorientation and induced fit upon RNA binding: solution structure and dynamics of ribosomal protein L11 from Thermotoga maritime. A European Journal of Chemical Biology, 6, 1611-1618. doi:10.1002/cbic.200500091
Datta, P.P., Sharma, M.R., Qi, L., Frank, J. and Agrawal, R.K. (2005) Interaction of the G' domain of elongation factor G and the C-terminal domain of ribosomal protein L7/L12 during translocation as revealed by cryo-EM. Molecular Cell, 20, 723-731. doi:10.1016/j.molcel.2005.10.028
Bowen, W.S., Van Dyke, N., Murgola, E.J., Lodmell, J.S. and Hill, W.E. (2005) Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain. Journal of Biological Chemistry, 280, 2934-2943. doi:10.1074/jbc.M407008200
Kavran, J.M. and Steitz, T.A. (2007) Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements. Journal of Biological Chemistry, 371, 1047-1059. doi:10.1016/j.jmb.2007.05.091
Todorova, R.T. and Saihara, Y. (2003) Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE. Molecular Biology Reports, 30, 113-119. doi:10.1023/A:1023991026045
Wilson, D.N., Schluenzen, F., Harms, J.M., Yoshida, T., Ohkubo, T., Albrecht, R., Buerger, J., Kobayashi, Y. and Fucini, P. (2005) X-ray crystallography study on ribosome recycling: The mechanism of binding and action of RRF on the 50S ribosomal subunit. The EMBO Journal, 24, 251-260. doi:10.1038/sj.emboj.7600525