Showing papers in "Annual Review of Biochemistry in 2000"
TL;DR: Detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins, which consists of two conserved components, a histidine protein kinase and a response regulator protein.
Abstract: ▪ Abstract Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regul...
3,406 citations
TL;DR: This review examines how the structures of these enzymes relate mechanistically to cyclooxygenase and peroxidase catalysis, and how differences in the structure of PGHS-2 confer on this isozyme differential sensitivity to COX-2 inhibitors.
Abstract: ▪ Abstract The prostaglandin endoperoxide H synthases-1 and 2 (PGHS-1 and PGHS-2; also cyclooxygenases-1 and 2, COX-1 and COX-2) catalyze the committed step in prostaglandin synthesis. PGHS-1 and 2 are of particular interest because they are the major targets of nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, and the new COX-2 inhibitors. Inhibition of the PGHSs with NSAIDs acutely reduces inflammation, pain, and fever, and long-term use of these drugs reduces fatal thrombotic events, as well as the development of colon cancer and Alzheimer's disease. In this review, we examine how the structures of these enzymes relate mechanistically to cyclooxygenase and peroxidase catalysis, and how differences in the structure of PGHS-2 confer on this isozyme differential sensitivity to COX-2 inhibitors. We further examine the evidence for independent signaling by PGHS-1 and PGHS-2, and the complex mechanisms for regulation of PGHS-2 gene expression.
2,780 citations
TL;DR: Comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fused mechanism.
Abstract: Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The structures of three conformations of the ectodomain of the 1968 Hong Kong influenza virus HA have been determined by X-ray crystallography: the single-chain precursor, HA0; the metastable neutral-pH conformation found on virus, and the fusion pH-induced conformation. These structures provide a framework for designing and interpreting the results of experiments on the activity of HA in receptor binding, the generation of emerging and reemerging epidemics, and membrane fusion during viral entry. Structures of HA in complex with sialic acid receptor analogs, together with binding experiments, provide details of these low-affinity interactions in terms of the sialic acid substituents recognized and the HA residues involved in recognition. Neutralizing antibody-binding sites surround the receptor-binding pocket on the membrane-distal surface of HA, and the structures of the complexes between neutralizing monoclonal Fabs and HA indicate possible neutralization mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in its structure primes HA for subsequent activation of membrane fusion at endosomal pH (Figure 1). Priming involves insertion of the fusion peptide into a charged pocket in the precursor; activation requires its extrusion towards the fusion target membrane, as the N terminus of a newly formed trimeric coiled coil, and repositioning of the C-terminal membrane anchor near the fusion peptide at the same end of a rod-shaped molecule. Comparison of this new HA conformation, which has been formed for membrane fusion, with the structures determined for other virus fusion glycoproteins suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fusion mechanism. Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion allows a similar conclusion.
2,629 citations
TL;DR: Current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis is reviewed, mainly prompted by the advent of whole genome sequencing and the availability of vast body of structural data.
Abstract: Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases. Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized. This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis.
1,270 citations
TL;DR: This review will highlight the important results that have emerged from high-resolution structural studies of protein tyrosine kinases and provide a molecular basis for understanding the mechanisms by which receptor and nonreceptor PTKs are regulated.
Abstract: ▪ Abstract Tyrosine phosphorylation is one of the key covalent modifications that occurs in multicellular organisms as a result of intercellular communication during embryogenesis and maintenance o...
1,156 citations
TL;DR: GAP activity can sharpen the termination of a signal upon removal of stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network.
Abstract: ▪ Abstract GTPase-activating proteins (GAPs) regulate heterotrimeric G proteins by increasing the rates at which their α subunits hydrolyze bound GTP and thus return to the inactive state. G protein GAPs act allosterically on Gα subunits, in contrast to GAPs for the Ras-like monomeric GTP-binding proteins. Although they do not contribute directly to the chemistry of GTP hydrolysis, G protein GAPs can accelerate hydrolysis >2000-fold. G protein GAPs include both effector proteins (phospholipase C-β, p115RhoGEF) and a growing family of regulators of G protein signaling (RGS proteins) that are found throughout the animal and fungal kingdoms. GAP activity can sharpen the termination of a signal upon removal of stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network. GAPs are regulated by various controls of their cellular concentrations, by complex interactions wit...
1,123 citations
TL;DR: The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems.
Abstract: ▪ Abstract In just a few short years, the chemical ligation of unprotected peptide segments in aqueous solution has established itself as the most practical method for the total synthesis of native proteins. A wide range of proteins has been prepared. These synthetic molecules have led to the elucidation of gene function, to the discovery of novel biology, and to the determination of new three-dimensional protein structures by both NMR and X-ray crystallography. The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems. Chemical protein synthesis has also enabled the systematic development of proteins with enhanced potency and specificity as candidate therapeutic agents.
1,087 citations
TL;DR: This article focuses on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding, and stresses the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.
Abstract: DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.
916 citations
TL;DR: The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubules function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal micro Tubulin behavior.
Abstract: ▪ Abstract Microtubules are polymers that are essential for, among other functions, cell transport and cell division in all eukaryotes. The regulation of the microtubule system includes transcription of different tubulin isotypes, folding of α/β-tubulin heterodimers, post-translation modification of tubulin, and nucleotide-based microtubule dynamics, as well as interaction with numerous microtubule-associated proteins that are themselves regulated. The result is the precise temporal and spatial pattern of microtubules that is observed throughout the cell cycle. The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubule function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal microtubule behavior. The combination of structural, genetic, biochemical, and biophysical data should soon give us a fuller understanding of the exquisite details in the regulation of the microtubule cytoskeleton.
771 citations
TL;DR: The lectin-monoglucosylated oligosaccharide interaction is one of the alternative ways by which cells retain improperly folded glycoproteins in the endoplasmic reticulum and increases folding efficiency, prevents premature glycoprotein oligomerization and degradation, and suppresses formation of non-native disulfide bonds.
Abstract: An unconventional mechanism for retaining improperly folded glycoproteins and facilitating acquisition of their native tertiary and quaternary structures operates in the endoplasmic reticulum. Recognition of folding glycoproteins by two resident lectins, membrane-bound calnexin and its soluble homolog, calreticulin, is mediated by protein-linked monoglucosylated oligosaccharides. These oligosaccharides contain glucose (Glc), mannose (Man), and N-acetylglucosamine (GlcNAc) in the general form Glc1Man7-9GlcNAc2. They are formed by glucosidase I- and II-catalyzed partial deglucosylation of the oligosaccharide transferred from dolichol diphosphate derivatives to Asn residues in nascent polypeptide chains (Glc3Man9GlcNAc2). Further deglucosylation of the oligosaccharides by glucosidase II liberates glycoproteins from their calnexin/calreticulin anchors. Monoglucosylated glycans are then recreated by the UDP-Glc:glycoprotein glucosyltransferase (GT), and thus recognized again by the lectins, only when linked to improperly folded protein moieties, as GT behaves as a sensor of glycoprotein conformations. The deglucosylation-reglucosylation cycle continues until proper folding is achieved. The lectin-monoglucosylated oligosaccharide interaction is one of the alternative ways by which cells retain improperly folded glycoproteins in the endoplasmic reticulum. Although it decreases the folding rate, it increases folding efficiency, prevents premature glycoprotein oligomerization and degradation, and suppresses formation of non-native disulfide bonds by hindering aggregation and thus allowing interaction of protein moieties of folding glycoproteins with classical chaperones and other proteins that assist in folding.
633 citations
TL;DR: It is concluded that the two-stage concept remains useful as an approach to simplifying discussions of stability, as a framework for folding concepts, and as a basis for understanding membrane protein evolution.
Abstract: ▪ Abstract Helical membrane protein folding and oligomerization can be usefully conceptualized as involving two energetically distinct stages—the formation and subsequent side-to-side association of independently stable transbilayer helices. The interactions of helices with the bilayer, with prosthetic groups, and with each other are examined in the context of recent evidence. We conclude that the two-stage concept remains useful as an approach to simplifying discussions of stability, as a framework for folding concepts, and as a basis for understanding membrane protein evolution.
TL;DR: Structural and mechanistic imperatives are becoming apparent as the assembly pathways and the coupling of multistep reactions catalyzed by these dauntingly complex molecular machines are unraveled.
Abstract: Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a number of the multifunctional enzyme systems responsible. The protein domains, for which the posttranslational machinery in the cell is highly specific, are crucially important, contributing to the processes of molecular recognition that define and protect the substrates and the catalytic intermediates. The domains have novel folds and move by virtue of conformationally flexible linker regions that tether them to other components of their respective multienzyme complexes. Structural and mechanistic imperatives are becoming apparent as the assembly pathways and the coupling of multistep reactions catalyzed by these dauntingly complex molecular machines are unraveled.
TL;DR: Models are presented that show how sequential NTP hydrolysis can lead to unidirectional and processive translocation and possible unwinding mechanisms based on the DNA exclusion model are proposed here, termed the wedge, torsional, and helix-destabilizing models.
Abstract: Helicases are motor proteins that couple the hydrolysis of nucleoside triphosphate (NTPase) to nucleic acid unwinding. The hexameric helicases have a characteristic ring-shaped structure, and all, except the eukaryotic minichromosomal maintenance (MCM) helicase, are homohexamers. Most of the 12 known hexameric helicases play a role in DNA replication, recombination, and transcription. A human genetic disorder, Bloom's syndrome, is associated with a defect in one member of the class of hexameric helicases. Significant progress has been made in understanding the biochemical properties, structures, and interactions of these helicases with DNA and nucleotides. Cooperativity in nucleotide binding was observed in many, and sequential NTPase catalysis has been observed in two proteins, gp4 of bacteriophage T7 and rho of Escherichia coli. The crystal structures of the oligomeric T7 gp4 helicase and the hexamer of RepA helicase show structural features that substantiate the observed cooperativity, and both are consistent with nucleotide binding at the subunit interface. Models are presented that show how sequential NTP hydrolysis can lead to unidirectional and processive translocation. Possible unwinding mechanisms based on the DNA exclusion model are proposed here, termed the wedge, torsional, and helix-destabilizing models.
TL;DR: The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc1 complexes and the chloroplast and cyanobacterial b6f complex.
Abstract: ▪ Abstract The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc 1 complexes and the chloroplast and cyanobacterial b 6 f complex. All these complexes couple electron transfer to proton translocation across a closed lipid bilayer membrane, conserving the free energy released by the oxidation-reduction process in the form of an electrochemical proton gradient across the membrane. Recent exciting developments include the application of site-directed mutagenesis to define the role of conserved residues, and the emergence over the past five years of X-ray structures for several mitochondrial complexes, and for two important domains of the b 6 f complex.
TL;DR: Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes, which functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription.
Abstract: ▪ Abstract Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interac...
TL;DR: Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties.
Abstract: Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.
TL;DR: The checkpoint mechanisms that function to preserve the integrity of the genome when the normal course of genome duplication is perturbed by factors that damage the DNA or inhibit DNA synthesis are described.
Abstract: ▪ Abstract The initiation of DNA replication in eukaryotic cells is tightly controlled to ensure that the genome is faithfully duplicated once each cell cycle. Genetic and biochemical studies in se...
TL;DR: Identification and characterization of the autophagic/cytoplasm-to-vacuole protein-targeting components have revealed the essential roles for various functional classes of proteins, including a novel protein conjugation system and the machinery for vesicle formation and fusion.
Abstract: The sequestration and delivery of cytoplasmic material to the yeast vacuole and mammalian lysosome require the dynamic mobilization of cellular membranes and specialized protein machinery. Under nutrient deprivation conditions, double-membrane vesicles form around bulk cytoplasmic cargo destined for degradation and recycling in the vacuole/lysosome. A similar process functions to remove excess organelles under vegetative conditions in which they are no longer needed. Biochemical, morphological, and molecular genetic studies in yeasts and mammalian cells have begun to elucidate the molecular details of this autophagy process. In addition, the overlap of macroautophagy with the process of pexophagy and with the biosynthetic cytoplasm-to-vacuole targeting pathway, which delivers the resident vacuolar hydrolase aminopeptidase I, indicates that these three pathways are related mechanistically. Identification and characterization of the autophagic/cytoplasm-to-vacuole protein-targeting components have revealed the essential roles for various functional classes of proteins, including a novel protein conjugation system and the machinery for vesicle formation and fusion.
TL;DR: This review summarizes spindle structure and highlights recent findings regarding the mechanisms and molecules involved in organizing microtubules into spindles.
Abstract: ▪ Abstract Chromosome segregation during mitosis and meiosis is driven by a complex superstructure called the spindle. Microtubules are the primary structural component of spindles, and spindle assembly and function are intimately linked to the intrinsic dynamics of microtubules. This review summarizes spindle structure and highlights recent findings regarding the mechanisms and molecules involved in organizing microtubules into spindles. In addition, mechanisms for chromosome movement and segregation are discussed.
TL;DR: This review discusses the current understanding of the three stages of large-scale structural changes of chromosomes in eukaryotic cells and identifies key components involved in these processes.
Abstract: ▪ Abstract The faithful segregation of genetic information requires highly orchestrated changes of chromosome structure during the mitotic cell cycle. The linkage between duplicated sister DNAs is established during S phase and maintained throughout G2 phase (cohesion). In early mitosis, dramatic structural changes occur to produce metaphase chromosomes, each consisting of a pair of compacted sister chromatids (condensation). At anaphase onset, a signal is produced to disrupt the linkage between sister chromatids (separation), allowing them to be pulled apart to opposite poles of the cell. This review discusses our current understanding of the three stages of large-scale structural changes of chromosomes in eukaryotic cells. Recent genetic and biochemical studies have identified key components involved in these processes and started to uncover hitherto unexpected functional links between mitotic chromosome dynamics and other important chromosome functions.
TL;DR: Genetic evidence shows that cryptochromes are circadian photoreceptors in Drosophila and Arabidopsis, raising the possibility that they may be universal circadian photoresceptors, and may provide new understanding of human diseases such as seasonal affective disorder and delayed sleep phase syndrome.
Abstract: ▪ Abstract Circadian rhythms are oscillations in the biochemical, physiological, and behavioral functions of organisms that occur with a periodicity of approximately 24 h. They are generated by a molecular clock that is synchronized with the solar day by environmental photic input. The cryptochromes are the mammalian circadian photoreceptors. They absorb light and transmit the electromagnetic signal to the molecular clock using a pterin and flavin adenine dinucleotide (FAD) as chromophore/cofactors, and are evolutionarily conserved and structurally related to the DNA repair enzyme photolyase. Humans and mice have two cryptochrome genes, CRY1 and CRY2, that are differentially expressed in the retina relative to the opsin-based visual photoreceptors. CRY1 is highly expressed with circadian periodicity in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Mutant mice lacking either Cry1 or Cry2 have impaired light induction of the clock gene mPer1 and have abnormally short or long intrinsi...
TL;DR: Development of improved transition-state analogs, refinements in immunization and screening protocols, and elaboration of general strategies for augmenting the efficiency of first-generation catalytic antibodies are identified as evident, but difficult, challenges for this field.
Abstract: Antibody molecules elicited with rationally designed transition-state analogs catalyze numerous reactions, including many that cannot be achieved by standard chemical methods. Although relatively primitive when compared with natural enzymes, these catalysts are valuable tools for probing the origins and evolution of biological catalysis. Mechanistic and structural analyses of representative antibody catalysts, generated with a variety of strategies for several different reaction types, suggest that their modest efficiency is a consequence of imperfect hapten design and indirect selection. Development of improved transition-state analogs, refinements in immunization and screening protocols, and elaboration of general strategies for augmenting the efficiency of first-generation catalytic antibodies are identified as evident, but difficult, challenges for this field. Rising to these challenges and more successfully integrating programmable design with the selective forces of biology will enhance our understanding of enzymatic catalysis. Further, it should yield useful protein catalysts for an enhanced range of practical applications in chemistry and biology.
TL;DR: The principal areas of progress are the identification of new peroxins, definition of protein-protein interactions among peroxin leading to the recognition of complexes involved in peroxisomal protein import, insight into the biogenesis ofperoxisome membrane proteins, and, of most importance, the elucidation of the role of many conservedperoxins in human disease.
Abstract: ▪ Abstract This review summarizes the progress made in our understanding of peroxisome biogenesis in the last few years, during which the functional roles of many of the 23 peroxins (proteins involved in peroxisomal protein import and peroxisome biogenesis) have become clearer. Previous reviews in the field have focussed on the metabolic functions of peroxisomes (1, 2), aspects of import/biogenesis (3, 4, 5, 6, 7), the role of peroxins in human disease (2, 8), and involvement of the endoplasmic reticulum in peroxisome membrane biogenesis (9, 10, 11) as well as the degradation of this organelle (5, 12). This review refers to some of the earlier work for the sake of introduction and continuity but deals primarily with the more recent progress. The principal areas of progress are the identification of new peroxins, definition of protein-protein interactions among peroxins leading to the recognition of complexes involved in peroxisomal protein import, insight into the biogenesis of peroxisomal membrane protei...
TL;DR: A modified paradigm of membrane fusion in which integral membrane proteins termed "SNAREs" can form stable complexes in cis (when on the same membrane) as well as in trans (when anchored to opposing membranes) is suggested.
Abstract: ▪ Abstract Homotypic (self) fusion of yeast vacuoles, which is essential for the low copy number of this organelle, uses catalytic elements similar to those used in heterotypic vesicular traffickin...
TL;DR: In this paper, the authors compared the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes.
Abstract: ▪ Abstract The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro–selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.
TL;DR: The properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover are discussed and several proteins required for mRNA decapping show interactions with the translation machinery and suggest possible mechanisms for the triggering of mRNA decappers.
Abstract: ▪ Abstract The process of mRNA turnover is a critical component of the regulation of gene expression. In the past few years a discrete set of pathways for the degradation of polyadenylated mRNAs in eukaryotic cells have been described. A major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which leads to a decapping reaction, thereby exposing the mRNA to rapid 5′ to 3′ exonucleolytic degradation. A critical step in this pathway is decapping, since it effectively terminates the existence of the mRNA and is the site of numerous control inputs. In this review, we discuss the properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover. A key point is that decapping appears to be controlled by access of the enzyme to the cap structure in a competition with the translation initiation complex. Strikingly, several proteins required for mRNA decapping show interactions with the translation machinery and suggest possible mechanisms for t...
TL;DR: The bypassing mechanism of T4 gene 60 is focused on in terms of take-off, scanning, and landing, which suggests that the strategy of translational bypassing may be more common than presently appreciated.
Abstract: ▪ Abstract Translational bypassing joins the information found within two disparate open reading frames into a single polypeptide chain. The underlying mechanism centers on the decoding properties of peptidyl-transfer RNA (tRNA) and involves three stages: take-off, scanning, and landing. In take-off, the peptidyl-tRNA/messenger RNA (mRNA) complex in the P site of the ribosome dissociates, and the mRNA begins to move through the ribosome. In scanning, the peptidyl-tRNA probes the mRNA sliding through the decoding center. In landing, the peptidyl-tRNA re-pairs with a codon with which it can form a stable interaction. Although few examples of genes are known that rely on translational bypassing to couple open reading frames, ribosomes appear to have an innate capacity for bypassing. This suggests that the strategy of translational bypassing may be more common than presently appreciated. The best characterized example of this phenomenon is T4 gene 60, in which a complex set of signals stimulates bypassing of ...
TL;DR: For almost 35 years the techniques of protein chemistry and molecular biology in studies of structural and conformational changes in the enzyme, the genes encoding the different polypeptides, subunit interactions, and assembly of the enzyme from six catalytic and six regulatory chains have been used.
Abstract: Following graduate training, which was disrupted by my changing schools and serving in the Navy in World War II, I arrived in Berkeley in 1948 as an instructor in the Biochemistry Department. Despite numerous academic reorganizations and a host of struggles over the University-imposed Loyalty Oath, dismissal of a faculty member because of political affiliations, free speech for students, and my resistance to mandatory retirement, I survived with the help of great graduate students, postdoctoral fellows, undergraduates, superb research assistants, and a supportive wife. Studies on structure of tobacco mosaic virus led to our investigating an ultracentrifuge anomaly and the construction of a synthetic boundary cell. In turn, this resulted in about 15 years of research on the ultracentrifuge and its application to the study of biological macromolecules. Among the latter, the discovery of large ribonucleoprotein complexes, now known as ribosomes, and chromatophores in photosynthetic microorganisms attracted t...