Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2007;35(4):1343-53.
doi: 10.1093/nar/gkl696. Epub 2007 Feb 1.

Release factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies

Gabriel Zoldák  1 , Lars Redecke, Dmitri I Svergun, Peter V Konarev, C Stefan Voertler, Holger Dobbek, Erik Sedlák, Mathias Sprinzl
Affiliations
Comparative Study

Release factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies

Gabriel Zoldák et al. Nucleic Acids Res. 2007.

Abstract

Prokaryotic class I release factors (RFs) respond to mRNA stop codons and terminate protein synthesis. They interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 A distant ribosomal centres. For this an elongated RF conformation, with partially unfolded core domains II.III.IV is required, which contrasts the known compact RF crystal structures. The crystal structure of Thermus thermophilus RF2 was determined and compared with solution structure of T. thermophilus and Escherichia coli RF2 by microcalorimetry, circular dichroism spectroscopy and small angle X-ray scattering. The structure of T. thermophilus RF2 in solution at 20 degrees C is predominantly compact like the crystal structure. Thermodynamic analysis point to an initial melting of domain I, which is independent from the melting of the core. The core domains II.III.IV melt cooperatively at the respective physiological temperatures for T. thermophilus and E. coli. Thermodynamic analyses and the X-ray scattering results for T. thermophilus RF2 in solution suggest that the compact conformation of RF2 resembles a physiological state in absence of the ribosome.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A) Structure model of Thermus thermophilus RF2 in two orientations with the colour coded domains I (blue), II (red), III (yellow) and IV (green). Tripeptide motifs are highlighted [GGQ and SP(F/Y); grey ovals] as well as the possible hinge regions for movement of domain III (black circles). (B) Overlay of the ribbon structures of E. coli (red) and T. thermophilus (green) RF2 structure models. (C) Model of an elongated, ribosomal bound E. coli RF2 as determined by cryo-electron microscopy (26,27). (D) Alignment of primary structures and hydropathy plot of E. coli and T. thermophilus RF2s.
Figure 1
Figure 1
(A) Structure model of Thermus thermophilus RF2 in two orientations with the colour coded domains I (blue), II (red), III (yellow) and IV (green). Tripeptide motifs are highlighted [GGQ and SP(F/Y); grey ovals] as well as the possible hinge regions for movement of domain III (black circles). (B) Overlay of the ribbon structures of E. coli (red) and T. thermophilus (green) RF2 structure models. (C) Model of an elongated, ribosomal bound E. coli RF2 as determined by cryo-electron microscopy (26,27). (D) Alignment of primary structures and hydropathy plot of E. coli and T. thermophilus RF2s.
Figure 2
Figure 2
Circular dichroism (CD) spectra of RF2 from E. coli and T. thermophilus in the far-UV region. CD spectra were measured in 2 mM sodium phosphate, pH 7.2, in a 1 mm path length cuvette. Concentrations of RF2 from E. coli (empty circles) and T. thermophilus (solid circles) were 2.95 and 1.4 μM, respectively. The fitted curves are shown by solid lines.
Figure 3
Figure 3
Guanidinium chloride induced unfolding of RF2 as monitored by the ellipticity at 220 nm. RF2 from E. coli (open circles) and T. thermophilus (solid circles) were measured at 1.5 μM protein concentration in 20 mM sodium cacodylate, pH 7.2 containing 300 mM NaCl and 1 mM β-mercaptoethanol at 20°C. Curves were generated by fitting the experimental data to Equation 3.
Figure 4
Figure 4
Temperature induced denaturation of E. coli and T. thermophilus RF2 monitored by CD and calorimetry. Upper panel: protein ellipticity in the far-UV region of CD at 220 nm. Scanning rate was 90 K/h, proteins concentration 1.5 μM in 20 mM sodium cacodylate, pH 7.2, 300 mM NaCl, and 1 mM β-mercapthoethanol. Ellipticity [Θ] is given in 10−3 deg cm2/dmol units. Lower panel: calorimetric scans of RFs (thick lines, E. coli and T. thermophilus) obtained upon subtraction of baselines. Second heating scans are shown as dashed (T. thermophilus) and dotted lines (E. coli). The scan rate was 90 K/min, the protein concentrations 24 μM. Curve fits of thermal denaturation based on the single and multiple two state processes are shown as solid lines. Inset: relative composition of ΔASA of interfaces between domain I and domains II·III·IV of RF2s from E. coli and T. thermophilus, respectively—polar amino acids (white box), nonpolar amino acids (grey box).
Figure 5
Figure 5
Small-angle X-ray scattering analysis of purified RF2 from T. thermophilus. (i) processed experimental SAXS pattern, dots with error bars; (ii) and (iii) scattering curves calculated from the closed X-ray structure of T. thermophilus RF2 determined and presented in this study and from the open X-ray structure of T. thermophilus RF2 when bound to a ribosome (PDB code 2B9M), respectively; (iv) scattering from a 78:22 mixture of the two conformations. The plot displays the logarithm of the relative scattering intensity as a function of the momentum transfer.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ogle J.M., Ramakrishnan V. Structural insights into translational fidelity. Annu. Rev. Biochem. 2005;74:129–177. - PubMed
    1. Schuwirth B.S., Borovinskaya M.A., Hau C.W., Zhang W., Vila-Sanjurjo A., Holton J.M., Cate J.H. Structures of the bacterial ribosome at 3.5 A resolution. Science. 2005;310:827–834. - PubMed
    1. Gao H., Sengupta J., Valle M., Korostelev A., Eswar N., Stagg S.M., Van Roey P., Agrawal R.K., Harvey S.C., Sali A., et al. Study of the structural dynamics of the Escherichia coli 70S ribosome using real-space refinement. Cell. 2003;113:789–801. - PubMed
    1. Frank J., Sengupta J., Gao H., Li W., Valle M., Zavialov A., Ehrenberg M. The role of tRNA as a molecular spring in decoding, accommodation, and peptidyl transfer. FEBS Lett. 2005;579:959–962. - PubMed
    1. Schmeing T.M., Huang K.S., Strobel S.A., Steitz T.A. An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA. Nature. 2005;438:520–524. - PubMed

Publication types

MeSH terms

Associated data