Temperature dependence for physical and functional reconstitution of 30S ribosomes of E. coli

Wireman, John W.; Sypherd, Paul S.

doi:10.1016/0006-291x(75)90548-3

Cited by 7 publications

(5 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Instead of having a relative sedimentation velocity of 30S, the particle containing the fragment sediments at approximately 28S (Fig 7), an assembly intermediate which has been previously observed (31,32).…”

Section: Discussionmentioning

confidence: 51%

Specific ribosomal RNA recognition by a fragment ofE. coliribosomal protein S4 missing the C-terminal 36 amino acid residues

Changchien

Craven

1985

Nucl Acids Res

View full text Add to dashboard Cite

We have previously investigated the role of the N-terminal region of ribosomal protein S4 to participate in 30S ribosome assembly and function (1-3). In this report we extend these studies to the two fragments produced by the chemical cleavage of protein S4 at the tryptophan residue 167. We find that the carboxyl terminal fragment (168-203) does not bind 16S RNA nor does it participate in assembly with the other 20 proteins from the 30S ribosome. In contrast, the larger fragment (1-167), does bind 16S RNA specifically. If the S4-fragment (1-167) is used to replace protein S4 in the complete 30S assembly reaction, all 20 of the other 30S proteins are incorporated. We conclude that the carboxyl terminal section of the protein S4 is not directly involved in binding 16S RNA or in the assembly of any of the other 30S proteins.

show abstract

Section: Discussionmentioning

confidence: 51%

Specific ribosomal RNA recognition by a fragment ofE. coliribosomal protein S4 missing the C-terminal 36 amino acid residues

Changchien

Craven

1985

Nucl Acids Res

View full text Add to dashboard Cite

show abstract

“…Therefore, we conclude that DnaK is not required for biosynthesis of ribosomal subunits, at least at 30°C, nor for the capabilities of ribosomal components to reconstitute active ribosomal subunits. Wireman and Sypherd (1975) detected disparity between the physical formation of 30S particles and the acquisition of activity when the reconstitution is performed with mature 16S rRNA: formation of 30S particles occurs at temperatures 15°C lower than the appearance of active subunits. Both features were reported to be improved by the DnaK system (Maki et al 2002), that is, reconstituted particles became more homogeneous as shown with sucrosegradient profiles, and tRNA binding occurred in the absence of a heat-activation step.…”

Section: Dnak Chaperones and Ribosome Assemblymentioning

confidence: 95%

DnaK-facilitated ribosome assembly inEscherichia colirevisited

Alix

Nierhaus

2003

RNA

View full text Add to dashboard Cite

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30°C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37°C implicates a direct or indirect role for DnaK in this process.

show abstract

“…Inactivation corresponds to conformational changes rather than to the loss of ribosomal components (Zamir et al, 1974). As a matter of fact, ab initio reconstitution of the 3OS subunit from isolated rRNA and ribosomal proteins exhibits too high energy requirements (Wireman and Sypherd, 1975) and would hardly explain the Mg2+ dependence of the rate-limiting process we measured (Figure 4b). No conformational change of the 50S subunit appears from the association kinetics we performed in the absence of the ammonium ion (Table II).…”

Section: Discussionmentioning

confidence: 78%

Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits

Favaudon¹,

François²

1976

Biochemistry

View full text Add to dashboard Cite

The magnesium dependence of the Escherichia coli ribosomal subunits association has been investigated by the stopped-flow technique using isolated 30S and 50S particles depleted of polyamines and any initiation factor. Binding of the fluorescent probe bis(8-anilino-1-naphthalenesulfonate) to the ribosomal proteins occurs through biphasic kinetics. A dark reaction corresponding to a very rapid, reversible complexation of the dye molecule is followed by a slow photochemical reaction that gives rise to irreversible addition of the probe. Only the 30S subparticle exhibits a magnesium-dependent conformational change from the kinetic analysis of the dark reaction. The 70S formation kinetics are limited by a conformational change of the 30S subunit if this particle is depleted of Mg2+ (1 mM Mg2+/50 mM K+), while its activated structure is restored by incubation with 8 mM Mg2+/50 mM K+. No rate-limiting conformation rearrangement of the 50S subunit could ever be evidenced. The Mg2+ dependence of the association kinetics of preactivated ribosomal particles is satisfactorily explained by electrostatic effects and/or formation of salt bridges, in agreement with the results of Wishnia and co-workers (Wishnia, A. Boussert, A., Graffe, M., Dessen, P., and Grunberg-Manago, M. (1975), J. Mol. Biol. 93, 499). Equilibrium studies indicate that the ribosomal preparations we used are of B type, according to Debey et al. (Debey, P., Hui Bon Hoa, G., Douzou, P., Godefroy-Colburn, T., Graffe, M., AND Grunberg-Manago, M. (1975) Biochemistry 14, 1553). The addition of spermidine results in a drastic fall of the need of Mg2+ for association, but it does not allow conversion of B-type particles into A-type ones at 25 degrees C. In addition to that, some 30S-bound spermidine appears to be involved directly in the coupling reaction.

show abstract

Temperature dependence for physical and functional reconstitution of 30S ribosomes of E. coli

Cited by 7 publications

References 14 publications

Specific ribosomal RNA recognition by a fragment ofE. coliribosomal protein S4 missing the C-terminal 36 amino acid residues

Specific ribosomal RNA recognition by a fragment ofE. coliribosomal protein S4 missing the C-terminal 36 amino acid residues

DnaK-facilitated ribosome assembly inEscherichia colirevisited

Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits

Contact Info

Product

Resources

About