Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7

Nowotny, Volker; Nierhaus, Knud H.

doi:10.1021/bi00418a057

Cited by 116 publications

(77 citation statements)

References 21 publications

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“…The proteins that bound transiently or partially in prebinding experiments were S5, S7, S9, S11, S13, and S19. A similar subset of proteins was found to bind transiently in a previous pulse-chase experiment designed to test binding stability in the presence of a chase (37). Unstable binding also occurred in the experiment with secondary proteins prebound, which showed no acceleration for primary or tertiary proteins.…”

Section: Efficiency Of Prebindingmentioning

confidence: 71%

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Bunner

Beck

Williamson

2010

Proc. Natl. Acad. Sci. U.S.A.

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The Escherichia coli 30S ribosomal subunit self-assembles in vitro in a hierarchical manner, with the RNA binding by proteins enabled by the prior binding of others under equilibrium conditions. Early 16S rRNA binding proteins also bind faster than late-binding proteins, but the specific causes for the slow binding of late proteins remain unclear. Previously, a pulse-chase monitored by quantitative mass spectrometry method was developed for monitoring 30S subunit assembly kinetics, and here a modified experimental scheme was used to probe kinetic cooperativity by including a step where subsets of ribosomal proteins bind and initiate assembly prior to the pulse-chase kinetics. In this work, 30S ribosomal subunit kinetic reconstitution experiments revealed that thermodynamic dependency does not always correlate with kinetic cooperativity. Some folding transitions that cause subsequent protein binding to be more energetically favorable do not result in faster protein binding. Although 3 0 domain primary protein S7 is required for RNA binding by both proteins S9 and S19, prior binding of S7 accelerates the binding of S9, but not S19, indicating there is an additional mechanistic step required for S19 to bind. Such data on kinetic cooperativity and the presence of multiphasic assembly kinetics reveal complexity in the assembly landscape that was previously hidden. mass spectrometry | RNA folding | RNA-protein interactions R ibosome biogenesis is a central cellular program that accounts for a significant fraction of the energy budget for rapidly growing bacteria, and is an essential process in all living cells. In eukaryotes, ribosome biogenesis in the nucleus requires hundreds of proteins (1, 2), whereas in bacteria the cytoplasmic assembly of ribosomes is facilitated by approximately 20 cofactors (3). Remarkably, the Escherichia coli 30S (4) and 50S (5) ribosomal subunits can be reconstituted in vitro, which has facilitated mechanistic studies on ribosome assembly. The 30S ribosomal subunit is a 900 kDa complex composed of 20 ribosomal proteins (r-proteins; S2, S3, ... S21) and a 1500-nucleotide rRNA (16S RNA). The 30S subunit is a well-characterized model system for studying macromolecular self-assembly in vitro (4), where protein binding occurs in a defined hierarchy and in a cooperative manner (6). The protein binding hierarchy was determined by a series of equilibrium reconstitution experiments that are summarized in the Nomura assembly map (6) (Fig. 1A). Primary proteins bind directly and independently to the 16S RNA, whereas secondary and tertiary proteins require prior binding of one or more proteins, respectively. The thermodynamic order of protein binding is generally consistent with kinetic binding data that show primary proteins binding fastest and tertiary proteins binding slowest (7,8). The assembly mechanism is also organized according to three structural domains, the 5 0 domain, the central domain, and the 3 0 domain (9), that can be reconstituted independently in vitro (10-12). Kinetic reconstitution...

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Section: Efficiency Of Prebindingmentioning

confidence: 71%

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Bunner

Beck

Williamson

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…To ensure each ribosomal subunit assembles completely, the ribosomal proteins bind to the 16S rRNA in a hierarchy that arises from protein-dependent conformational changes in the 16S rRNA (Held et al 1974;Held and Nomura 1975;Nowotny and Nierhaus 1988). Biochemical and biophysical studies have confirmed the cooperative binding of proteins to the central and 3…”

Section: Introductionmentioning

confidence: 99%

Differential effects of ribosomal proteins and Mg²⁺ ions on a conformational switch during 30S ribosome 5′-domain assembly

Abeysirigunawardena

Woodson

2015

RNA

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Ribosomal protein S4 nucleates assembly of the 30S ribosome 5 ′ and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg 2+ ions and other 5 ′ -domain proteins. Equilibrium titrations of Cy3-labeled 5 ′ -domain RNA with Cy5-labeled protein S4 showed that Mg 2+ ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg 2+ ions alone.

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“…Mutations that disrupt this interaction affect translational fidelity in Escherichia coli, leading to an increased capacity for frameshifting and readthrough (Robert and Brakier-Gingras 2003). In addition, E. coli rpS7 initiates assembly of the 30S subunit by binding to 16S rRNA (Nowotny and Nierhaus 1988;Fredrick et al 2000;Grondek and Culver 2004). In contrast, very little is known about the function of rpS7's eukaryotic rpS5 counterpart.…”

Section: Introductionmentioning

confidence: 99%

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Galkin

Bentley

Gupta

et al. 2007

RNA

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Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI ;3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and À1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.

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Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7

Cited by 116 publications

References 21 publications

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Differential effects of ribosomal proteins and Mg²⁺ ions on a conformational switch during 30S ribosome 5′-domain assembly

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

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Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7

Cited by 116 publications

References 21 publications

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

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Differential effects of ribosomal proteins and Mg²⁺ ions on a conformational switch during 30S ribosome 5′-domain assembly