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Chapter 25 : Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G

Authors: Marina V. Rodnina1, Andreas Savelsbergh1, Dagmar Mohr1, Natalia B. Matassova1, Wolfgang Wintermeyer1, Tillmann Pape2
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Affiliations: 1: Institute of Molecular Biology, University of Witten / Herdecke, 58448 Witten, Germany; 2: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom
Content Type: Monograph
Publication Year: 2000

Category: Microbial Genetics and Molecular Biology

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Abstract:

This chapter concentrates on pre-steady-state kinetic work, and provides evidence on how much the interpretation of kinetic results in molecular-mechanistic terms owes to structural information obtained from crystallography and cryo-electron microscopy. Our studies of elongation factor (EF-Tu) function address two main issues: (i) the elucidation of the reaction pathway to identify intermediate steps of A-site binding and (ii) the quantitative evaluation of the pathway in order to understand specificity. Based on measured rates of GTP hydrolysis and peptide bond formation, Thompson and colleagues proposed that the rate of GTP hydrolysis by EF-Tu is independent of the tRNA, thereby providing an internal kinetic standard for translational accuracy. Binding of thiostrepton to the 1070 region of 23S rRNA interferes with translocation, as it strongly inhibits P release, translocation, and subsequent turnover of EF-G; in contrast, EF-G binding and GTP hydrolysis are not affected. The GTPase activities of EF-Tu and EF-G intrinsically are very low and are strongly enhanced on the ribosome. Recently, the ability of isolated L12 protein to stimulate GTP hydrolysis by either EF-Tu or EF-G, has been studied. In the fusidic acid-stabilized complex of EF-G with the ribosome, the α- sarcin stem is close to position 196 in the G domain of EF-G, which lies just above the GTP binding site, while the α-sarcin loop region is in the vicinity of position 650 in domain 5 of the factor.

Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
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Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G
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Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
/content/10.1128/9781555818142.chap25.fig25-1
Figure 1

Mechanism of EF-Tu-dependent binding of aa-tRNA to the ribosomal A site. EF-Tu is depicted in three conformations: the GTP-bound form (gray), the transient GTPase-activated form on the ribosome (white), and the GDP-bound form (gray) that dissociates from the ribosome. The kinetic parameters are summarized in ( Table 1 .

10.1128/9781555818142/fig25-1_thmb.gif
10.1128/9781555818142/fig25-1.gif
Image of Figure 1
Figure 1

Mechanism of EF-Tu-dependent binding of aa-tRNA to the ribosomal A site. EF-Tu is depicted in three conformations: the GTP-bound form (gray), the transient GTPase-activated form on the ribosome (white), and the GDP-bound form (gray) that dissociates from the ribosome. The kinetic parameters are summarized in ( Table 1 .

Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
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Figure 2

Reaction scheme of translocation as discussed in the text. EF-G is depicted in three conformations: the GTP-bound form, an intermediate GDP-bound form on the ribosome, and the GDP-bound form that dissociates from the ribosome. The transition state of the ribosome, formed in step 3, is symbolized by an altered conformation of the small ribosomal subunit. A, P (P*), and E denote the tRNA binding sites on the two subunits and are indicated when occupied. The kinetic parameters are summarized in Table 3 .

10.1128/9781555818142/fig25-2_thmb.gif
10.1128/9781555818142/fig25-2.gif
Image of Figure 2
Figure 2

Reaction scheme of translocation as discussed in the text. EF-G is depicted in three conformations: the GTP-bound form, an intermediate GDP-bound form on the ribosome, and the GDP-bound form that dissociates from the ribosome. The transition state of the ribosome, formed in step 3, is symbolized by an altered conformation of the small ribosomal subunit. A, P (P*), and E denote the tRNA binding sites on the two subunits and are indicated when occupied. The kinetic parameters are summarized in Table 3 .

Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
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Tables

Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G
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Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
/content/10.1128/9781555818142.chap25.tab25-1
Table 1

Elemental rate constants of cognate, near-cognate, and noncognate aa-tRNA binding to the A site

Generic image for table
Table 1

Elemental rate constants of cognate, near-cognate, and noncognate aa-tRNA binding to the A site

Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
/content/10.1128/9781555818142.chap25.tab25-2
Table 2

Effect of paromomycin on the elemental rate constants of A-site binding of near-cognate aa-tRNA

Generic image for table
Table 2

Effect of paromomycin on the elemental rate constants of A-site binding of near-cognate aa-tRNA

Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25
/content/10.1128/9781555818142.chap25.tab25-3
Table 3

Kinetic parameters of translocation

Generic image for table
Table 3

Kinetic parameters of translocation

Citation: Rodnina M, Savelsbergh A, Mohr D, Matassova N, Wintermeyer W, Pape T. 2000. Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G, p 301-318. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch25

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