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.
Subject(s)
Mitochondria/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructure , Animals , Binding Sites , Cattle , Cryoelectron Microscopy , Cytoplasm/metabolism , GTP-Binding Proteins/metabolism , Image Processing, Computer-Assisted , Liver/metabolism , Protein Conformation , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/metabolism , Ribosomal Proteins/metabolismABSTRACT
The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes).
Subject(s)
Leishmania/genetics , Mitochondria/chemistry , Ribosomes/chemistry , Animals , Cryoelectron Microscopy , Mitochondria/metabolism , Models, Molecular , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructureABSTRACT
Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.
Subject(s)
Bacterial Proteins/chemistry , RNA-Binding Proteins/chemistry , Ribosomal Proteins/chemistry , Ribosome Subunits, Small, Bacterial/metabolism , Thermus thermophilus/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Binding Sites , Cryoelectron Microscopy , Models, Molecular , Protein Structure, Tertiary , RNA, Bacterial/metabolism , RNA, Ribosomal, 16S/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Ribosomal Proteins/physiologyABSTRACT
Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.
Subject(s)
Escherichia coli Proteins/metabolism , GTP-Binding Proteins/metabolism , Protein Subunits/metabolism , RNA, Ribosomal, 16S/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Binding Sites , Cryoelectron Microscopy , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/ultrastructure , Models, Molecular , Multiprotein Complexes , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , RNA, Ribosomal, 16S/ultrastructure , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/ultrastructure , Thermus thermophilus/genetics , Thermus thermophilus/metabolismABSTRACT
After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosome-recycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by approximately 13 A) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA.
Subject(s)
Escherichia coli/chemistry , Proteins/chemistry , Proteins/physiology , Ribosomes/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , Escherichia coli/genetics , Models, Molecular , Molecular Conformation , Protein Binding , Protein Structure, Tertiary , Proteins/genetics , Proteins/metabolism , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomal Proteins , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
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.
