Mobile domains in ribosomes revealed by proton nuclear magnetic resonance.

Cowgill, Cynthia; Nichols, Brenda G.; Kenny, James W.; Butler, Peter D.; Bradbury, E. M.; Traut, Robert R.

doi:10.1016/s0021-9258(17)42543-9

Cited by 61 publications

(14 citation statements)

References 25 publications

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“…along the L7/12 stalk (Stoffler & Stoffler-Meilicke, 1986); thus, the cross-linking reagent would have to span a distance of approximately 70 A. There is, however, evidence that one dimer of L7/12 may not be located in the stalk (Boublik et al, 1975; McKuskie-Olson et al, 1985;Traut et al, 1986) and that the stalk may be flexible (Cowgill et al, 1984), two findings which could easily explain the cross-link L5-L7/12. The fact that multiple cross-links with L7/12 are found with most cross-linking reagents may be an indication for the flexibility of the stalk.…”

Section: Discussionmentioning

confidence: 99%

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

Walleczek

Martin²,

Redl³

et al. 1989

Biochemistry

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We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.

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Section: Discussionmentioning

confidence: 99%

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

Walleczek

Martin²,

Redl³

et al. 1989

Biochemistry

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“…L7/L12 is considered to be comprised of two distinct, organized, structural domains: a helical N-terminal domain (Mo ¨ller & Maassen, 1986;Tsurugi & Mitsui, 1991;Bocharov et al, 1996), residues 1-36, responsible for dimer formation (Gudkov & Behlke, 1978;Gudkov et al, 1995) and for binding of the L7/L12 dimer to L10 in the 50S ribosomal subunit, and a globular C-terminal domain (Leijonmarck & Liljas, 1987), residues 53-120, responsible for interaction with factors (Van Agthaven et al, 1975;Sommer et al, 1985). These domains are separated by a putatively disordered "hinge" region, residues 35-52 (Gudkov et al, 1982;Cowgill et al, 1984;Leijonmarck & Liljas, 1987;Bushuev et al, 1989).…”

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confidence: 99%

“…Mutants of L7/L12 lacking this hinge region are no longer competent to promote ribosome activity (Kirsebom et al, 1986;Gudkov et al, 1991;Traut et al, 1993;Oleinikov et al, 1993a). Flexibility of L7/L12 has also been inferred from proton NMR (Cowgill et al, 1984;Bushuev et al, 1984Bushuev et al, , 1989, ESR (Tritton, 1978), and electron microscopy (Verschoor et al, 1986). These studies suggest that L7/L12 does not behave as a rigid rod but rather can display flexibility and relative motion between the C-and N-terminal domains.…”

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confidence: 99%

Rotational and Conformational Dynamics of Escherichia coli Ribosomal Protein L7/L12

et al. 1996

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Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

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“…The location of both dimers of L7/L12 on the ribosome is unclear, and the determination is made more difficult insofar as the two dimers may have different locations (MSller et al, 1983;Olson et al, 1986;Zantema et al, 1982) and because one or both dimers are mobile (Cowgill et al, 1984;Gudkov et al, 1982;Tritton, 1978) and may undergo major conformational changes during the ribosome cycle. Cross-links between L7/L12 and proteins distant from the L7/L12 stalk have been identified (Redl et al, 1989;Traut et al, 1983).…”

mentioning

confidence: 99%

The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide

Zecherle¹,

Oleinikov²,

Traut³

1992

Biochemistry

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All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis. The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propionamide . Properties of this reagent are described. It was first radiolabeled with 125I and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89. Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89. Irradiation of the reconstituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components. Reductive cleavage of the disulfide cross-link resulted in transfer of the 125I label from L7/L12Cys89 to the other cross-linked components. Two radiolabeled proteins were identified, L11 and L10. The location of both of these proteins is well established to be at the base of the L7/L12 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors. The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain. The cross-linking method with radiolabeled N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.

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Mobile domains in ribosomes revealed by proton nuclear magnetic resonance.

Cited by 61 publications

References 25 publications

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

Rotational and Conformational Dynamics of Escherichia coli Ribosomal Protein L7/L12

The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide

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