Mapping structural differences between 30S ribosomal subunit assembly intermediates

Holmes, Kristi L.; Culver, Gloria M.

doi:10.1038/nsmb719

Cited by 63 publications

(90 citation statements)

References 37 publications

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“…These structures correspond to assembly intermediates lacking mainly proteins of the 30S head, giving structural support for the proposed 5Ј to 3Ј directionality of the assembly process (48,49). The magnesium depletion appears to destabilize the 30S head relative to the 30S body and platform, leading to an apparently reverse process of weakening 3Ј domain interactions relative to 5Ј interactions.…”

Section: Inactive Conformation Of 30s Mimics Ribosome Biogenesismentioning

confidence: 56%

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

Boehringer

O′Farrell

Rife

et al. 2012

Journal of Biological Chemistry

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Background:The RNA methyltransferase KsgA is required for small ribosomal subunit maturation. Results: Cryo-electron microscopy reveals the structure of the near mature ribosomal subunit in complex with KsgA. Conclusion: We suggest that KsgA controls conformational changes in the ribosomal RNA required for final subunit maturation. Significance: The significance is understanding the remodeling and processing of ribosomal RNA by protein factors that are required for ribosome biogenesis.

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Section: Inactive Conformation Of 30s Mimics Ribosome Biogenesismentioning

confidence: 56%

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

Boehringer

O′Farrell

Rife

et al. 2012

Journal of Biological Chemistry

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“…In addition to providing information on intact ribosomes, this approach should be applicable to examining ribosome dissociation processes, subunit associations and conformational changes associated with the binding of external ribosomal factors. Another particular useful extension of this method would be the investigation of other RNP complexes for which little high-resolution structural data exists, such as the ribosomal subunit assembly intermediates [45,46,51]. Thus, while this present study does not advance directly our understanding of the E. coli or T. thermophilus ribosomes, it does demonstrate the feasibility of a relatively straight-forward and rapid mass spectrometric approach for limited proteolysis of large RNP complexes.…”

Section: Discussionmentioning

confidence: 92%

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Suh

Pourshahian

Limbach

2007

J. Am. Soc. Mass Spectrom.

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An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30-60 min of incubation provide insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.The ribosome, found in all organisms, is the subcellular organelle that performs the activity of protein synthesis. Prokaryotic ribosomes, which sediment at 70S, have a molecular mass of approximately 2.5 ×10 6 Da and consist of two subunits, the 50S and the 30S. Each subunit has a unique number of proteins and ribosomal RNAs (rRNAs). Eukaryotic ribosomes, which sediment at 80S, are substantially larger and more complex than their prokaryotic counterparts. Two subunits, the 60S and 40S, together contain at least 78 unique proteins and 4 rRNAs. The small subunit binds messenger RNA (mRNA) and mediates the interactions between mRNA and transfer RNAs (tRNAs). The larger subunit catalyzes peptide-bond formation. During the initiation phase of protein synthesis, the two subunits behave independently, assembling into complete ribosomes only when elongation is about to begin.A fundamental prerequisite for understanding the biochemical interactions occurring within the ribosome during protein synthesis is a detailed knowledge of the structure of this † Current address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10021 * To whom correspondence should be addressed. Phone (513) 556-1871, Fax (513) Email: Pat.Limbach@uc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

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“…Such slow refolding from kinetic traps has frequently been observed in studies of other large RNAs (18)(19)(20). Three lines of evidence led to the hypothesis that the 3 0 domain has a stronger tendency to fall into kinetic traps than the two other domains: The 3 0 domain assembles slowest at all temperatures (8), and many 3 0 domain proteins are clustered around the mRNA decoding site (9), which undergoes conformational changes during the late stages of assembly (21). In addition, chemical modification data from various stages of 30S reconstitution (22) have shown reactivity enhancements to occur in the 3 0 domain in the late stages of assembly, which suggests that nonnative interactions within the 3 0 domain are unmade.…”

mentioning

confidence: 88%

“…In the case of S19 prebinding, kinetic cooperativity was observed where there was no thermodynamic dependency, suggesting S19 may direct assembly through channels not evident from thermodynamic data. Although this was surprising, it has been previously noted that S19 likely plays a greater role than other 3 0 domain secondary proteins in 16S rRNA conformational changes during the late stages of assembly (21). In addition, S19 is known to interact with the ribosome biogenesis factor RimM (34), so those two proteins together may facilitate efficient 3 0 domain assembly in vivo.…”

Section: Fig 4 (A)-(b)mentioning

confidence: 99%

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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Mapping structural differences between 30S ribosomal subunit assembly intermediates

Cited by 63 publications

References 37 publications

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

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

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