The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform

Rajendra K Agrawal - One of the best experts on this subject based on the ideXlab platform.

  • Structures of the human Mitochondrial Ribosome recycling complexes reveal distinct mechanisms of recycling and antibiotic resistance
    2020
    Co-Authors: Ravi K. Koripella, Ayush Deep, Ekansh K. Agrawal, Pooja Keshavan, Nilesh K. Banavali, Rajendra K Agrawal
    Abstract:

    Ribosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Mitochondrial Ribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt) are two proteins with mitochondria-specific additional sequences that catalyze the recycling step in human mitochondria. We have determined high-resolution cryo-EM structures of the human 55S Mitochondrial Ribosome (mitoRibosome) in complex with RRFmt, and the mitoribosomal large 39S subunit in complex with both RRFmt and EF-G2mt. In addition, we have captured the structure of a short-lived intermediate state of the 55S*RRFmt*EF-G2mt complex. These structures clarify the role of a mitochondria-specific segment of RRFmt in mitoRibosome recycling, identify the structural distinctions between the two isoforms of EF-Gmt that confer their functional specificity, capture recycling-specific conformational changes in the L7/L12 stalk-base region, and suggest a distinct mechanistic sequence of events in mitoRibosome recycling. Furthermore, biochemical and structural assessments of the sensitivity of EF-G2mt to the antibiotic fusidic acid reveals that the molecular mechanism of antibiotic resistance for EF-G2mt is markedly different from that exhibited by Mitochondrial elongation factor EF-G1mt, suggesting that these two homologous Mitochondrial proteins have evolved diversely to negate the effect of a bacterial antibiotics.

  • structural insights into unique features of the human Mitochondrial Ribosome recycling
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Ravi K. Koripella, Manjuli R Sharma, Rajendra K Agrawal, Pooja Keshavan, Paul Risteff
    Abstract:

    Mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation (ATP generation). Despite their common ancestry with bacteria, the composition and structure of the human mitoRibosome and its translational factors are significantly different from those of their bacterial counterparts. The mammalian mitoRibosome recycling factor (RRFmt) carries a mito-specific N terminus extension (NTE), which is necessary for the function of RRFmt. Here we present a 3.9-A resolution cryo-electron microscopic (cryo-EM) structure of the human 55S mitoRibosome-RRFmt complex, which reveals α-helix and loop structures for the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoRibosome. These include ribosomal RNA segments that constitute the peptidyl transferase center (PTC) and those that connect PTC with the GTPase-associated center and with mitoribosomal proteins L16 and L27. Our structure reveals the presence of a tRNA in the pe/E position and a rotation of the small mitoribosomal subunit on RRFmt binding. In addition, we observe an interaction between the pe/E tRNA and a mito-specific protein, mL64. These findings help understand the unique features of mitoRibosome recycling.

  • Structural insights into unique features of the human Mitochondrial Ribosome recycling
    2018
    Co-Authors: Ravi K. Koripella, Manjuli R Sharma, Pooja Keshavan, Paul Risteff, Rajendra K Agrawal
    Abstract:

    Mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation or energy (ATP) generation. Despite their proposed bacterial origin, the composition and structure of the human mitoRibosome and its translational factors are dramatically different from their bacterial counterparts. The mammalian mitoRibosome recycling factor (RRFmt) carries a mito-specific N-terminus extension (NTE), which is necessary for the function of RRFmt. Here we present a 3.7 Angstrom resolution cryo-EM structure of the human 55S mitoRibosome-RRFmt complex, which reveals α-helix and loop structures for a portion of the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoRibosome. These include ribosomal RNAs segments that constitute the peptidyl transferase center (PTC), those that connect PTC with the GTPase-associated center, and with multiple mitoribosomal proteins. Our structure also reveals novel conformational changes in mitoRibosome due to RRFmt binding. Together, these findings help understand the unique features of mitoRibosome recycling.

  • The 55S mammalian Mitochondrial Ribosome and its tRNA-exit region
    Biochimie, 2015
    Co-Authors: Prem Singh Kaushal, Manjuli R Sharma, Rajendra K Agrawal
    Abstract:

    Mitochondria carry their own genetic material and gene-expression machinery, including Ribosomes, which are responsible for synthesizing polypeptides that form essential components of the complexes involved in oxidative phosphorylation (or ATP generation) for the eukaryotic cell. Mitochondrial Ribosomes (mitoRibosomes) are quite divergent from cytoplasmic Ribosomes in both composition and structure even as their main functional cores, such as the mRNA decoding and peptidyl transferase sites, are highly conserved. Remarkable progress has been made recently towards understanding the structure of mitoRibosomes, by obtaining high-resolution cryo-electron microscopic (cryo-EM) maps. These studies confirm previous structural findings that had revealed that a significant reduction in size of ribosomal RNAs has caused topological changes in some of the functionally relevant regions, including the transfer RNA (tRNA)-binding sites and the nascent polypeptide-exit tunnel, within the structure of the mammalian mitoRibosome. In addition, these studies provide unprecedented detailed views of the molecular architecture of those regions. In this review, we summarize the current state of knowledge of the structure of the mammalian mitoRibosome and describe the molecular environment of its tRNA-exit region.

  • cryo em structure of the small subunit of the mammalian Mitochondrial Ribosome
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Prem Singh Kaushal, Linda L Spremulli, Emdadul Haque, Manjuli R Sharma, T M Booth, Chang Shung Tung, Karissa Y Sanbonmatsu, Rajendra K Agrawal
    Abstract:

    The mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoRibosome contains significantly smaller rRNAs and a large mass of Mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoRibosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoRibosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5′ leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoRibosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges.

Linda L Spremulli - One of the best experts on this subject based on the ideXlab platform.

  • structures of the human Mitochondrial Ribosome bound to ef g1 reveal distinct features of Mitochondrial translation elongation
    Nature Communications, 2020
    Co-Authors: Ravi K. Koripella, Linda L Spremulli, Manjuli R Sharma, Partha P Datta, Pooja Keshavan, Nilesh K. Banavali, Kalpana Bhargava, Prem Singh Kaushal
    Abstract:

    The mammalian Mitochondrial Ribosome (mitoRibosome) and its associated translational factors have evolved to accommodate greater participation of proteins in Mitochondrial translation. Here we present the 2.68–3.96 A cryo-EM structures of the human 55S mitoRibosome in complex with the human Mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in Mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoRibosome onto the inner-Mitochondrial membrane. Translation within mitochondria is carried out by specialized mitoRibosomes and translational factors. Here the authors describe cryo-EM structures of the human Mitochondrial translation elongation factor G1 in complex with human mitoRibosomes, revealing distinct mechanism that include conformational changes at the polypeptide exit tunnel.

  • cryo em structure of the small subunit of the mammalian Mitochondrial Ribosome
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Prem Singh Kaushal, Linda L Spremulli, Emdadul Haque, Manjuli R Sharma, T M Booth, Chang Shung Tung, Karissa Y Sanbonmatsu, Rajendra K Agrawal
    Abstract:

    The mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoRibosome contains significantly smaller rRNAs and a large mass of Mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoRibosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoRibosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5′ leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoRibosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges.

  • Current Views of the Structure of the Mammalian Mitochondrial Ribosome
    Israel Journal of Chemistry, 2010
    Co-Authors: Emine C. Koc, Emdadul Haque, Linda L Spremulli
    Abstract:

    Mammalian mitochondria synthesize polypeptides crucial for energy generation using Ribosomes with a number of unique features. These Ribosomes are very protein rich and have very truncated ribosomal RNAs. The bulk of the mammalian Mitochondrial Ribosome is composed of proteins, only about half of which are homologs of ribosomal proteins found in other translational systems. A number of distinctive features are found in these Ribosomes. Among these is a gate-like structure that allows entrance of the primarily leaderless mRNAs that characterize this system. The exit tunnel of the large subunit is also quite unusual and includes a site in which the nascent peptide is visible to solvent prior to the normal exit site. Further, this region of the Mitochondrial Ribosome is dominated by ribosomal proteins rather than rRNA and is involved in the interaction of the Ribosome with the inner membrane where all of the translation products are ultimately located. The proteins of the Mitochondrial Ribosome appear to play a number of important roles in the cell in addition to their function in protein biosynthesis, including roles in apoptosis and in cell cycle control.

  • proteomics and electron microscopic characterization of the unusual Mitochondrial Ribosome related 45s complex in leishmania tarentolae
    Molecular and Biochemical Parasitology, 2007
    Co-Authors: Dmitri A. Maslov, Linda L Spremulli, Manjuli R Sharma, Rajendra K Agrawal, Kalpana Bhargava, Domenick Grasso, Arnold M Falick, Carol E Parker, Larry Simpson
    Abstract:

    Abstract A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant Mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein–protein (coiled-coil, Rhodanese) or protein–RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania Mitochondrial Ribosome is associated with an additional protein mass. The biological role of these particles is not known.

  • structure of the mammalian Mitochondrial Ribosome reveals an expanded functional role for its component proteins
    Cell, 2003
    Co-Authors: Manjuli R Sharma, Linda L Spremulli, Emine C. Koc, Partha P Datta, T M Booth, Rajendra K Agrawal
    Abstract:

    The Mitochondrial Ribosome is responsible for the biosynthesis of protein components crucial to the generation of ATP in the eukaryotic cell. Because the protein:RNA ratio in the Mitochondrial Ribosome (approximately 69:approximately 31) is the inverse of that of its prokaryotic counterpart (approximately 33:approximately 67), it was thought that the additional and/or larger proteins of the Mitochondrial Ribosome must compensate for the shortened rRNAs. Here, we present a three-dimensional cryo-electron microscopic map of the mammalian Mitochondrial 55S Ribosome carrying a tRNA at its P site, and we find that instead, many of the proteins occupy new positions in the Ribosome. Furthermore, unlike cytoplasmic Ribosomes, the Mitochondrial Ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique Mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.

Manjuli R Sharma - One of the best experts on this subject based on the ideXlab platform.

  • structures of the human Mitochondrial Ribosome bound to ef g1 reveal distinct features of Mitochondrial translation elongation
    Nature Communications, 2020
    Co-Authors: Ravi K. Koripella, Linda L Spremulli, Manjuli R Sharma, Partha P Datta, Pooja Keshavan, Nilesh K. Banavali, Kalpana Bhargava, Prem Singh Kaushal
    Abstract:

    The mammalian Mitochondrial Ribosome (mitoRibosome) and its associated translational factors have evolved to accommodate greater participation of proteins in Mitochondrial translation. Here we present the 2.68–3.96 A cryo-EM structures of the human 55S mitoRibosome in complex with the human Mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in Mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoRibosome onto the inner-Mitochondrial membrane. Translation within mitochondria is carried out by specialized mitoRibosomes and translational factors. Here the authors describe cryo-EM structures of the human Mitochondrial translation elongation factor G1 in complex with human mitoRibosomes, revealing distinct mechanism that include conformational changes at the polypeptide exit tunnel.

  • structural insights into unique features of the human Mitochondrial Ribosome recycling
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Ravi K. Koripella, Manjuli R Sharma, Rajendra K Agrawal, Pooja Keshavan, Paul Risteff
    Abstract:

    Mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation (ATP generation). Despite their common ancestry with bacteria, the composition and structure of the human mitoRibosome and its translational factors are significantly different from those of their bacterial counterparts. The mammalian mitoRibosome recycling factor (RRFmt) carries a mito-specific N terminus extension (NTE), which is necessary for the function of RRFmt. Here we present a 3.9-A resolution cryo-electron microscopic (cryo-EM) structure of the human 55S mitoRibosome-RRFmt complex, which reveals α-helix and loop structures for the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoRibosome. These include ribosomal RNA segments that constitute the peptidyl transferase center (PTC) and those that connect PTC with the GTPase-associated center and with mitoribosomal proteins L16 and L27. Our structure reveals the presence of a tRNA in the pe/E position and a rotation of the small mitoribosomal subunit on RRFmt binding. In addition, we observe an interaction between the pe/E tRNA and a mito-specific protein, mL64. These findings help understand the unique features of mitoRibosome recycling.

  • Structural insights into unique features of the human Mitochondrial Ribosome recycling
    2018
    Co-Authors: Ravi K. Koripella, Manjuli R Sharma, Pooja Keshavan, Paul Risteff, Rajendra K Agrawal
    Abstract:

    Mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation or energy (ATP) generation. Despite their proposed bacterial origin, the composition and structure of the human mitoRibosome and its translational factors are dramatically different from their bacterial counterparts. The mammalian mitoRibosome recycling factor (RRFmt) carries a mito-specific N-terminus extension (NTE), which is necessary for the function of RRFmt. Here we present a 3.7 Angstrom resolution cryo-EM structure of the human 55S mitoRibosome-RRFmt complex, which reveals α-helix and loop structures for a portion of the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoRibosome. These include ribosomal RNAs segments that constitute the peptidyl transferase center (PTC), those that connect PTC with the GTPase-associated center, and with multiple mitoribosomal proteins. Our structure also reveals novel conformational changes in mitoRibosome due to RRFmt binding. Together, these findings help understand the unique features of mitoRibosome recycling.

  • The 55S mammalian Mitochondrial Ribosome and its tRNA-exit region
    Biochimie, 2015
    Co-Authors: Prem Singh Kaushal, Manjuli R Sharma, Rajendra K Agrawal
    Abstract:

    Mitochondria carry their own genetic material and gene-expression machinery, including Ribosomes, which are responsible for synthesizing polypeptides that form essential components of the complexes involved in oxidative phosphorylation (or ATP generation) for the eukaryotic cell. Mitochondrial Ribosomes (mitoRibosomes) are quite divergent from cytoplasmic Ribosomes in both composition and structure even as their main functional cores, such as the mRNA decoding and peptidyl transferase sites, are highly conserved. Remarkable progress has been made recently towards understanding the structure of mitoRibosomes, by obtaining high-resolution cryo-electron microscopic (cryo-EM) maps. These studies confirm previous structural findings that had revealed that a significant reduction in size of ribosomal RNAs has caused topological changes in some of the functionally relevant regions, including the transfer RNA (tRNA)-binding sites and the nascent polypeptide-exit tunnel, within the structure of the mammalian mitoRibosome. In addition, these studies provide unprecedented detailed views of the molecular architecture of those regions. In this review, we summarize the current state of knowledge of the structure of the mammalian mitoRibosome and describe the molecular environment of its tRNA-exit region.

  • cryo em structure of the small subunit of the mammalian Mitochondrial Ribosome
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Prem Singh Kaushal, Linda L Spremulli, Emdadul Haque, Manjuli R Sharma, T M Booth, Chang Shung Tung, Karissa Y Sanbonmatsu, Rajendra K Agrawal
    Abstract:

    The mammalian Mitochondrial Ribosomes (mitoRibosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoRibosome contains significantly smaller rRNAs and a large mass of Mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoRibosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoRibosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5′ leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoRibosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges.

Emine C. Koc - One of the best experts on this subject based on the ideXlab platform.

  • Current Views of the Structure of the Mammalian Mitochondrial Ribosome
    Israel Journal of Chemistry, 2010
    Co-Authors: Emine C. Koc, Emdadul Haque, Linda L Spremulli
    Abstract:

    Mammalian mitochondria synthesize polypeptides crucial for energy generation using Ribosomes with a number of unique features. These Ribosomes are very protein rich and have very truncated ribosomal RNAs. The bulk of the mammalian Mitochondrial Ribosome is composed of proteins, only about half of which are homologs of ribosomal proteins found in other translational systems. A number of distinctive features are found in these Ribosomes. Among these is a gate-like structure that allows entrance of the primarily leaderless mRNAs that characterize this system. The exit tunnel of the large subunit is also quite unusual and includes a site in which the nascent peptide is visible to solvent prior to the normal exit site. Further, this region of the Mitochondrial Ribosome is dominated by ribosomal proteins rather than rRNA and is involved in the interaction of the Ribosome with the inner membrane where all of the translation products are ultimately located. The proteins of the Mitochondrial Ribosome appear to play a number of important roles in the cell in addition to their function in protein biosynthesis, including roles in apoptosis and in cell cycle control.

  • phosphorylated proteins of the mammalian Mitochondrial Ribosome implications in protein synthesis
    Journal of Proteome Research, 2009
    Co-Authors: Jennifer L Miller, Huseyin Cimen, Hasan Koc, Emine C. Koc
    Abstract:

    Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 Mitochondrially encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of Mitochondrial ribosomal proteins and their role(s) in Ribosome function. In this study, we have identified the Mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated Ribosomes that were phosphorylated in vitro by kinases PKA, PKCdelta, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of Mitochondrial translation machinery by phosphorylation and other post-translational modifications.

  • structure of the mammalian Mitochondrial Ribosome reveals an expanded functional role for its component proteins
    Cell, 2003
    Co-Authors: Manjuli R Sharma, Linda L Spremulli, Emine C. Koc, Partha P Datta, T M Booth, Rajendra K Agrawal
    Abstract:

    The Mitochondrial Ribosome is responsible for the biosynthesis of protein components crucial to the generation of ATP in the eukaryotic cell. Because the protein:RNA ratio in the Mitochondrial Ribosome (approximately 69:approximately 31) is the inverse of that of its prokaryotic counterpart (approximately 33:approximately 67), it was thought that the additional and/or larger proteins of the Mitochondrial Ribosome must compensate for the shortened rRNAs. Here, we present a three-dimensional cryo-electron microscopic map of the mammalian Mitochondrial 55S Ribosome carrying a tRNA at its P site, and we find that instead, many of the proteins occupy new positions in the Ribosome. Furthermore, unlike cytoplasmic Ribosomes, the Mitochondrial Ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique Mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.

  • The Large Subunit of the Mammalian Mitochondrial Ribosome ANALYSIS OF THE COMPLEMENT OF RIBOSOMAL PROTEINS PRESENT
    The Journal of biological chemistry, 2001
    Co-Authors: Emine C. Koc, William Burkhart, Kevin Blackburn, Arthur Moseley, Mary B. Moyer, Daniela M. Schlatzer, Linda L Spremulli
    Abstract:

    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, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae Mitochondrial Ribosomes. No Mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to Mitochondrial Ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic Ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 Mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.

  • the small subunit of the mammalian Mitochondrial Ribosome identification of the full complement of ribosomal proteins present
    Journal of Biological Chemistry, 2001
    Co-Authors: Emine C. Koc, William Burkhart, Kevin Blackburn, Arthur Moseley, Linda L Spremulli
    Abstract:

    Identification of all the protein components of the small subunit (28 S) of the mammalian Mitochondrial Ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human Mitochondrial Ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae Mitochondrial Ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster Mitochondrial Ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No Mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian Mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to Mitochondrial Ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic Ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the Mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.

Joanna Rorbach - One of the best experts on this subject based on the ideXlab platform.

  • Structures of the human Mitochondrial Ribosome in native states of assembly.
    Nature structural & molecular biology, 2017
    Co-Authors: Alan Brown, Joanna Rorbach, Alexey Amunts, Sorbhi Rathore, Dari Kimanius, Shintaro Aibara, Xiao Chen Bai, V Ramakrishnan
    Abstract:

    Mammalian Mitochondrial Ribosomes (mitoRibosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial Ribosome. These differences make the assembly of mitoRibosomes more complex than the assembly of bacterial Ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ∼3-A resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoRibosomes. Furthermore, the structures redefine the Ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

  • The human RNA-binding protein RBFA promotes the maturation of the Mitochondrial Ribosome.
    The Biochemical journal, 2017
    Co-Authors: Agata Rozanska, Joanna Rorbach, Ricarda Richter-dennerlein, Fei Gao, Richard J. Lewis, Zofia M.a. Chrzanowska-lightowlers, Robert N. Lightowlers
    Abstract:

    Accurate assembly and maturation of human Mitochondrial Ribosomes is essential for synthesis of the 13 polypeptides encoded by the Mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (Mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human Ribosome-binding factor A (RBFA) is a Mitochondrial RNA-binding protein that exerts crucial roles in mitoRibosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoRibosome formation.

  • MRM2 and MRM3 are involved in biogenesis of the large subunit of the Mitochondrial Ribosome
    Molecular biology of the cell, 2014
    Co-Authors: Joanna Rorbach, Payam A. Gammage, Pierre Boesch, Thomas J. Nicholls, Sarah F. Pearce, Dipali Patel, Andreas Hauser, Fabiana Perocchi, Michal Minczuk
    Abstract:

    Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human Mitochondrial 16S rRNA and its role in biogenesis of the mitoRibosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished Mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the Mitochondrial Ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human Mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the Mitochondrial Ribosome.

  • C7orf30 is necessary for biogenesis of the large subunit of the Mitochondrial Ribosome
    Nucleic acids research, 2012
    Co-Authors: Joanna Rorbach, Payam A. Gammage, Michal Minczuk
    Abstract:

    Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Here, we characterize a novel human protein, C7orf30 that contributes critically to Mitochondrial translation and specifically associates with the large subunit of the Mitochondrial Ribosome (mt-LSU). Inactivation of C7orf30 in human cells by RNA interference results in respiratory incompetence owing to reduced Mitochondrial translation rates without any appreciable effects on the steady-state levels of Mitochondrial mRNAs and rRNAs. Ineffective translation in C7orf30-depleted cells or cells overexpressing a dominant-negative mutant of the protein results from aberrant assembly of mt-LSU and consequently reduced formation of the monosome. These findings lead us to propose that C7orf30 is a human assembly and/or stability factor involved in the biogenesis of the large subunit of the Mitochondrial Ribosome.

  • a functional peptidyl trna hydrolase ict1 has been recruited into the human Mitochondrial Ribosome
    The EMBO Journal, 2010
    Co-Authors: Ricarda Richter, Aleksandra Pajak, Robert N. Lightowlers, Martijn A. Huynen, Joanna Rorbach, Paul M Smith, Hans J C T Wessels, Jan A M Smeitink, Zofia M A Chrzanowskalightowlers
    Abstract:

    Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative Mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 Mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential Mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoRibosome. Release-factor assays show that although ICT1 has retained its Ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to Ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoRibosomes.