ABSTRACT
In the process of protein synthesis, the translating ribosomes of eukaryotic cells form polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic polyribosomes are circles (circular polyribosomes) and two-stranded formations (so-called double-row polyribosomes). The "long" (i.e. heavy loaded) polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular ("collapsed rings"), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in polyribosomes by marking them with gold nanoparticles (3'-end) and initiating 40S subunits (5'-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in polyribosomes and mutual orientation of ribosomes in them has shown that the double-row polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.
Subject(s)
Eukaryota/genetics , Polyribosomes/chemistry , RNA, Messenger/chemistry , Eukaryota/chemistry , Eukaryota/metabolism , Humans , Nucleic Acid Conformation , Polyribosomes/genetics , Polyribosomes/metabolism , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Messenger/metabolismABSTRACT
The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNA(fMet) to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation.
Subject(s)
Bacteria/genetics , Peptide Chain Initiation, Translational , Protein Biosynthesis , Bacteria/metabolism , Models, Molecular , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Nucleic Acid Conformation , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer, Met/chemistry , Ribosome Subunits, Small, Bacterial/chemistry , Ribosome Subunits, Small, Bacterial/genetics , Ribosome Subunits, Small, Bacterial/metabolismABSTRACT
The human nuclear receptor of retinoic acid hRARgamma is a ligand-dependent transcription regulator. The presence of a completely ordered dodecyl-alpha-D-maltoside molecule in the crystal structure of the hRARgamma ligand-binding domain (LBD) refined at 1. 3 A resolution is reported. The non-ionic detergent is required for stabilization and crystallization of the hRARgamma LBD and mediates a crystal contact in the region where coactivator proteins bind. Its dodecyl moiety is buried in a hydrophobic channel, whereas the maltoside head group is hydrogen bonded to water molecules and polar residue side chains.
Subject(s)
Detergents/chemistry , Receptors, Retinoic Acid/chemistry , Crystallization , Humans , Models, Molecular , Protein Conformation , Retinoic Acid Receptor gammaABSTRACT
The human retinoic acid receptor (hRAR) belongs to the family of nuclear receptors that regulate transcription in a ligand-dependent way. The isotypes RARalpha,beta and gamma are distinct pharmacological targets for retinoids that are involved in the treatment of various skin diseases and cancers, in particular breast cancer and acute promyelocytic leukemia. Therefore, synthetic retinoids have been developed aiming at isotype selectivity and reduced side-effects. We report the crystal structures of three complexes of the hRARgamma ligand-binding domain (LBD) bound to agonist retinoids that possess selectivity either for RARgamma (BMS184394) or for RARbeta/gamma (CD564), or that are potent for all RAR-isotypes (panagonist BMS181156). The high resolution data (1.3-1. 5 A) provide a description at the atomic level of the ligand pocket revealing the molecular determinants for the different degrees of ligand selectivity. The comparison of the complexes of the chemically closely related retinoids BMS184394 and CD564 shows that the side-chain of Met272 adopts different conformations depending on the presence of a hydrogen bond between its sulfur atom and the ligand. This accounts for their different isotype selectivity. On the other hand, the difference between the pan- and the RARbeta, gamma-selective agonist is probably due to a steric discrimination at the level of the 2-naphthoic acid moiety of CD564. Based on this study, we propose a model for a complex with the RARgamma-specific agonist CD666 that shows the possible applications for structure-based drug design of RAR isotype-selective retinoids.
Subject(s)
Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Retinoids/chemistry , Retinoids/metabolism , Binding Sites , Crystallography, X-Ray , Drug Design , Humans , Hydrogen Bonding , Ligands , Models, Molecular , Protein Isoforms/agonists , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Tertiary , Receptors, Retinoic Acid/agonists , Retinoids/pharmacology , Structure-Activity Relationship , Substrate Specificity , Retinoic Acid Receptor gammaABSTRACT
Nuclear hormone receptors are transcription factors regulated by lipophilic ligands. These hormones bind to their nuclear receptor targets using an induced fit mechanism that triggers a large conformational change and generates the proper surface for the binding of protein coactivators. The molecular details of the various steps of this activation process or its inhibition by antagonists are now understood for several nuclear receptors.
Subject(s)
Hormones/chemistry , Hormones/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Animals , Ligands , Models, Molecular , Protein ConformationABSTRACT
The human retinoic acid receptor (hRAR) is a member of the nuclear receptor superfamily that regulates the transcription of target genes in a ligand-dependent manner. The three hRAR isotypes are targets for retinoids that are used in the treatment of various diseases, including breast cancer and skin diseases. Drug efficiency and safety depend on the pharmacological activity of enantiomers, which can differ because of the chiral environment generated by the target. We report the crystal structures of the hRARgamma ligand-binding domain bound to two enantiomers, the active BMS270394 and the inactive BMS270395, solved at 1.6 A and 1.7 A resolution, respectively. The crystal structures reveal that in both enantiomers, the hydroxyl moiety attached to the chiral center forms a hydrogen bond to the Met-272 sulfur atom, thus imposing a conformation of BMS270395 that differs significantly from that observed for BMS270394 and other known retinoids. BMS270395 adopts an energetically unfavorable conformation, accounting for its inactivity; in contrast, the conformation of BMS270394 is close to an energy minimum. Our high-resolution data allow rationalization of enantiomer discrimination by the receptor and provide a model system for the pharmacological properties of enantiomeric pairs.
