Characterization of functionally active subribosomal particles from
            <i>Thermus aquaticus</i>

Khaitovich, Philipp; Mankin, Alexander S.; Green, Rachel; Lancaster, Laura; Noller, Harry F.

doi:10.1073/pnas.96.1.85

Cited by 91 publications

(58 citation statements)

References 42 publications

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“…Even under these conditions, no difference in the mass spectral results was seen as compared to the data obtained at 1500 min (data not shown). The significantly greater number of large subunit as compared to small subunit r-proteins that were resistant to limited proteolysis using trypsin is a reflection of the different organization and r-protein:rRNA mass ratio of the two subunits [38,39]. The mass spectral data limited to those proteins remaining intact after limited proteolysis with trypsin is summarized in Figure 3.…”

Section: Limited Proteolysis and Ribosome Integritymentioning

confidence: 99%

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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Section: Limited Proteolysis and Ribosome Integritymentioning

confidence: 99%

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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“…Ribosomal L18p/L5e family proteins are one of the three 5 S rRNA-binding proteins in prokaryotic and cytoplasmic ribosomes (41). L18 is also one of the eight essential ribosomal proteins required to form active subribosomal particles of Thermus aquaticus ribosomes containing both 23 and 5 S rRNAs (31). Recently, the arginine-rich N-terminal portion of Pyrococcus furiosus, L18 was shown to be involved in 5 S rRNA binding (30).…”

Section: Discussionmentioning

confidence: 99%

“…This region corresponds to the C-terminal end of prokaryotic and chloroplast L28s. The C-terminal 128 residues of human MRP-L28 was previously identified as a "human melanoma-associated antigen" (Swiss-Prot O13084) that is recognized by certain melanoma-specific tumor-infiltrating lymphocytes (31). Extensive EST data base searches indicate that this data base entry does not represent the complete open reading frame for this protein.…”

Section: Icatmentioning

confidence: 99%

The Large Subunit of the Mammalian Mitochondrial Ribosome

Koc¹,

Burkhart²,

Blackburn³

et al. 2001

Journal of Biological Chemistry

244

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Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3 , L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, Mammalian mitochondria are responsible for the synthesis of 13 proteins localized in the inner membrane. These proteins are components of the oligomeric complexes essential for oxidative phosphorylation and, hence, for the synthesis of about 90% of the ATP in eukaryotic organisms. The 55 S mammalian mitochondrial ribosomes consists of small (28 S) and large (39 S) subunits (1). In contrast to bacterial ribosomes which are about 65% RNA, mammalian mitochondrial ribosomes are only 33% RNA. The low percentage of RNA in these ribosomes reflects a reduction in the size of the rRNA and a compensating increase in the number of ribosomal proteins. For example, the small subunit of the mammalian mitochondrial ribosome contains a 12 S rRNA (about 950 nucleotides) and an estimated 29 proteins (2). In contrast, the Escherichia coli 30 S subunit has a 16 S rRNA (1542 nucleotides in length) and 21 proteins (3). The large subunit of the mammalian mitochondrial ribosome contains a 16 S rRNA (about 1560 nucleotides) and about 50 proteins (4, 5).The identification of proteins in mammalian mitochondrial ribosomes has been challenging due to their low abundance. Recently 60 mammalian mitochondrial ribosomal proteins, 31 proteins from the large subunit and 29 proteins from the small subunit, have been characterized by different laboratories (2, 6 -14). The identification of these proteins used two approaches. The traditional approach was to separate the proteins on two-dimensional gels or high performance liquid chromatography followed by sequence analysis using Edman chemistry or mass spectrometry (MS). More recently, proteins present in the 28 S subunit have been characterized by proteolytic digestion of whole subunits. Sequence information on the peptides present in this complex mixture was obtained by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS).1 This strategy allowed the identification of 28 proteins of the small subunit including 14 proteins that had not previously been identified (2). In the present study, we have extended this approach to the 39 S subunit. In addition to direct analysis of 39 S digests by LC/MS, aliquots of the total digest were fractionated prior to reversed-phase LC/MS analysis to maximize the number of peptides sequenced. In the first approach, a portion of the total digest was fractionated by affinity selection ...

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“…It also shows that the 30 S proteins S13, S15, and the 50S proteins L2, L5, L14, L19 are involved in intersubunit bridges, while the 30 S proteins S9, S13, and the 50 S proteins L1, L5, L33 interact with tRNAs, suggesting that they could participate in the translocation step (12). Studies in solution also point to a role for the ribosomal proteins, such as an involvement in mRNA binding for S1 (14 -16), a participation in peptidyl transferase activity for L2 (17)(18)(19) and the prevention of mRNA slippage for L9 (20).…”

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confidence: 99%

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

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In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.The ribosome is the cellular machinery responsible for protein synthesis in all living organisms. The elucidation of the crystal structure of the prokaryotic ribosome has led to a major progress in understanding its function (1-7). Analysis of the ribosome structure combined with a wealth of biochemical data clearly showed that ribosomal RNA (rRNA) is the key player in the functions of the ribosome, and it is currently assumed that the main task of the ribosomal proteins is to help and stabilize rRNA folding and to facilitate conformational changes in rRNA (8 -10). However, a growing list of examples suggests that ribosomal proteins directly participate to protein synthesis. The x-ray structure of the ribosome reveals that S12 is part of the decoding site (Refs. 11 and 12, reviewed in Ref. 13). It also shows that the 30 S proteins S13, S15, and the 50S proteins L2, L5, L14, L19 are involved in intersubunit bridges, while the 30 S proteins S9, S13, and the 50 S proteins L1, L5, L33 interact with tRNAs, suggesting that they could participate in the translocation step (12). Studies in solution also point to a role for the ribosomal proteins, such as an involvement in mRNA binding for S1 (14 -16), a participation in peptidyl transferase activity for L2 (17-19) and the prevention of mRNA slippage for L9 (20).Several protein-protein interactions were identified in the crystal structure of the 30 S subunit (21, 22) but, so far, only the interaction between proteins S4 and S5 has been directly related to a particular ribosome function, that is the control of translational fidelity. Indeed, mutations that disrupt the S4-S5 interaction decrease translational accuracy (23,24). Among the other protein-protein inter...

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Characterization of functionally active subribosomal particles from Thermus aquaticus

Cited by 91 publications

References 42 publications

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

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

The Large Subunit of the Mammalian Mitochondrial Ribosome

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

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