The inactivation and reactivation of ribosomal-peptidyl transferase of E. coli

Miskin, Ruth; Zamir, Ada; Elson, David

doi:10.1016/0006-291x(68)90330-6

Cited by 83 publications

(37 citation statements)

References 8 publications

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“…The various monovalent cations were effective in maintaining the 60-5 subunit activity although not all to the same extent. Thus, liver subunits behaved differently to bacterial subunits, which were both reversibly inactivated when depleted of K+ [1,2]. Our findings on the activity of the standard 40-S subunits in the absence of monocations and also their ability to bind [14C]phenylalanyl-tRNA a t low MgC1, concentration differ from those of Castles et al [lo].…”

Section: Discussioncontrasting

confidence: 76%

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Ribosomal Subunits from Rat Liver

Reboud

Buisson

Reboud

1972

European Journal of Biochemistry

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Large subunits from rat liver ribosomes, dialyzed against monovalent cation-free buffer, were completely inactive in polyphenlyalanine synthesis and failed to reassociate with standard 403 subunits. Small subunits dialyzed under exactly the same conditions kept about of their activity in poly(U)-directed [l4C]phenyla1anyl-tRNA binding or protein synthesis and could reassociate with standard 60-S subunits. The nature of the monovalent cation present in low concentrations in the dialysis buffer was important in determining the activity of the 60-5, but not of the 40-5 subunits: K+, Rb+ and NH,+ were more effective while Cs+, Li+ and Na+ were less effective.The various monovalent cations could be used a t higher concentrations to prepare active subunits, except for Cs+ and Li+ which selectively inactivated the 60-5 subunits while modifying their conformation. Experiments with bivalent cations also showed the cationic content of the medium to be more critical for maintaining the functional form of 60-S than of 4043 subunits.The monovalent cations were thought until recently to have no special function in ribosomal structure except of interfering with Mga+ binding.However, it now appears that certain monovalent cations are required in order to prepare active ribosomes or ribosomal subunits from bacteria [1,2] and eucaryotic cells [3-51. Mg2+ cannot replace these monovalent cations.We have shown recently [6,7] that rat liver ribosomes could be completely dissociated into subunits using monovalent cations other than K+, that is Li+, Na+, Rb+, Cs+ and NH,+. The activity of the subunits in polyphenylalanine synthesis depended on the nature and concentration of the cation. As in these experiments ribosomal subunits had not been isolated, we could not completely exclude the possibilit

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Section: Discussioncontrasting

confidence: 76%

“…However, it now appears that certain monovalent cations are required in order to prepare active ribosomes or ribosomal subunits from bacteria [1,2] and eucaryotic cells [3-51. Mg2+ cannot replace these monovalent cations.…”

mentioning

confidence: 99%

Ribosomal Subunits from Rat Liver

Reboud

Buisson

Reboud

1972

European Journal of Biochemistry

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“…11,41 Despite the PTC high conservation, variability of its conformation resulting from alterations of parameters such pH, temperature and ion concentration, has been suggested, based on biochemical findings, over three decades ago. [42][43][44] These observations were confirmed recently by several methods, including chemical probing 45 and cryo-electron microscopy studies, investigating the effect of buffer composition on tRNA-ribosome interactons. 46 Consistently, conformational differences were observed by comparing the available crystal structures that were determined under various conditions.…”

Section: Regulation Discrimination and Signaling Subunit Associationmentioning

confidence: 54%

“…8,9,11 Correlation between the ribosomal functional state and the conformation of its PTC has been suggested, based on functional, biochemical and genetic evidence. 42,45 Consistently, disorder in the H50S crystal structure, 2 observed despite the high level of order within the ribosomal core, could be correlated with the environment of the crystal, which is far from that leading to efficient protein biosynthesis. 21 It is conceivable that disorder of functionally relevant components is induced by the ribosome itself, aimed at avoiding unproductive subunit association and substrate binding, when the conditions are not suitable for efficient operation.…”

Section: Regulation Discrimination and Signaling Subunit Associationmentioning

confidence: 96%

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

Zarivach

Bashan

Berisio

et al. 2004

J of Physical Organic Chem

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High-resolution structures of both ribosomal subunits revealed that most stages of protein biosynthesis, including decoding of genetic information, are navigated and controlled by the elaborate ribosomal architecturaldesign. Remote interactions govern accurate substrate alignment within a flexible active-site pocket [peptidyl transferase center (PTC)], and spatial considerations, due mainly to a universal mobile nucleotide, U2585, ensure proper chirality by interfering with D-amino-acids incorporation. tRNA translocation involves two correlated motions: overall mRNA/tRNA (messenger and transfer RNA) shift, and a rotation of the tRNA single-stranded aminoacylated-3 0 end around the bond connecting it with the tRNA helical-regions. This bond coincides with an axis passing through a sizable symmetry-related region, identified around the PTC in all large-subunit crystal structures. Propelled by a bulged universal nucleotide, A2602, positioned at the two-fold symmetry axis, and guided by a ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry optimal for peptide-bond formation and in geometry ensuring nascent proteins entrance into their exit tunnel. Hence, confirming that ribosomes contribute positional rather than chemical catalysis, and that peptide bond formation is concurrent with A-to P-site tRNA passage. Connecting between the PTC, the decoding center, the tRNA entrance and exit points, the symmetry-related region can transfer intra-ribosomal signals between remote functional locations, guaranteeing smooth processivity of amino acids polymerization. Ribosomal proteins are involved in accurate substrate placement (L16), discrimination and signal transmission (L22) and protein biosynthesis regulation (CTC). Residing on the exit tunnel walls near its entrance, and stretching to its opening, protein-L22 can mediate ribosome response to cellular regulatory signals, since it can swing across the tunnel, causing gating and elongation arrest. Each of the protein CTC domains has a defined task. The N-terminal domain stabilizes the intersubunit-bridge confining the A-site-tRNA entrance. The middle domain protects the bridge conformation at elevated temperatures. The C-terminal domain can undergo substantial conformational rearrangements upon substrate binding, indicating CTC participation in biosynthesiscontrol under stressful conditions.

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“…The ribosomes (tight couples) from E. coli MRE-600 were isolated in a sucrose density gradient in a zonal rotor [ 1 ] and activated before the following experiment [2]; pA-Met+-f was synthesized by the imidazole method [3]. For aminoacylation, a total preparation of tRNA from E. coli B was used.…”

Section: Preparation Of Ribosomes and Substratesmentioning

confidence: 99%