Adenylate/uridylate-rich elements (AREs) are found in the 3' untranslated region (UTR) of many messenger RNAs (mRNAs) that code for proto-oncogenes, nuclear transcription factors and cytokines. They represent the most common determinant of RNA stability in mammalian cells. Moreover, ARE-directed mRNA degradation is influenced by many exogenous factors, including phorbol esters, calcium ionophores, cytokines and transcription inhibitors. These observations suggest that AREs play a critical role in the regulation of gene expression during cell growth and differentiation, and in the immune response.
If you are studying cytochrome c oxidase in Paracoccus because it is more convenient than doing experiments with the homologous enzyme from human mitochondria, you are exploiting the conservation of the basic biochemical machinery in living organisms through billions of years of evolution. Such evolutionary connections are evident in the results of sequence database searches using programs such as BLAST or FASTA. On one hand, the power of these database searches derives from a simple rule of thumb: sequence identity above 25% (higher for shorter sequences) implies a conserved three-dimensional fold and, very likely, conserved biological function. However, the reverse is not true: protein families diverge with few specific constraints on the amino acid sequence. For example, comparison of ancient branches of globins (myoglobin, haemoglobin, leghaemoglobin) reveals that only one position in the chain requires an invariant amino acid, yet the same haem-binding function and a conserved three-dimensional fold are retained. Increasingly, ancient evolutionary relationships, which are no longer evident from staring at sequences alone, are being revealed by structure-structure comparisons. When successful, structure comparison searches in databases can lead to a considerable information gain through precise prediction of protein function (e.g. identification of the active site in barley endochitinase via reference to lysozymes) and unification of several protein families into a functional superfamily (e.g. DNA polymerase beta with kanamycin nucleotidyltransferase and others). The recent surge in newly solved three-dimensional protein structures has whetted the appetite for systematic structure-structure comparison with the potential for fascinating evolutionary discoveries. To make tools for such discoveries generally available, the European Molecular Biology Laboratory is now providing Internet access to the Dali method for protein structure comparison. The services include a database of precalculated structural neighbours for all public structures and an e-mail server for searches with newly solved ones.
There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts. In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose). DNA repair occurs after dissociation of modified PARP from DNA strand breaks. Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair. Possible roles for poly(ADP-ribose) synthesis are discussed.
During Drosophila development, the Polycomb and trithorax group genes (Pc-G and trx-G) are required to maintain stable expression patterns for the clustered homeotic genes (HOM-C). Several lines of evidence suggest that Pc-G and trx-G exert their effects through interaction with, or modification of, chromatin. Using the recently published sequence of the Pc-G protein Polycomblike (Pcl) as a probe in sequence similarity searches, we found that a zinc-finger-like motif occurs twice in Pcl and three times in trx and HRX. This motif has a unique Cys sub(4)-His-Cys sub(3) pattern, spanning approximately 50-80 residues. The pattern of conservation, which includes additional conserved positions, is clearly distinct from two similarly sized motifs that also occur in nuclear regulatory proteins; the Cys sub(3)-His-Cys sub(4) RING finger and the Cys sub(2)-His-Cys sub(5) LIM domain. Schindler et al. noted this Cys-rich motif in two closely related plant homeodomain proteins, HAT3.1 and HOX1A, and called it the PHD finger.
The Ca2+-binding protein calmodulin binds to and activates several cellular enzymes in response to a rise in Ca2+ concentration. It binds certain basic amphiphilic helices within these enzymes, which also act as autoinhibitory domains. The modulation of the binding equilibrium of these helices between intramolecular (inhibition) and intermolecular (activation) sites forms a focal point for crosstalk between various signalling pathways.
The addition of glucose to cells of the yeast Saccharomyces cerevisiae triggers a variety of regulatory phenomena. Initial glucose metabolism is required for the induction of most of them. Mutants deficient in both glucose-induced signalling and the control of initial glucose metabolism have a defect in the trehalose-6-phosphate synthase catalytic subunit of the trehalose synthase complex. This finding has raised novel questions about the control of glucose influx into glycolysis in yeast and its connection to the glucose-sensing mechanism. This dual function of the trehalose-6-phosphate synthase subunit has been found in several yeast species, suggesting that this control system might be widespread in fungi and possibly also in other organisms.
Intracellular proteins involved in regulatory and signal transduction processes frequently contain regions of localized similarity to otherwise unrelated proteins. These homology domains usually mediate the specific interaction with other proteins or subcellular structures. Only the relatively well-conserved examples are detectable by conventional pairwise sequence comparison techniques. The discovery of the more divergent functional domains requires application of specialized methods with increased sensitivity, together with a rigorous statistical evaluation of the results. Within the family of the forkhead-type transcription factors, sequence conservation is usually limited to the actual DNA-binding domain. Starting from the observation of an additional conserved region found in a subset of forkhead-family proteins, we applied the generalized sequence profile method to find further instances of this domain, which we named the forkhead-associated (FHA) domain. We were able to detect FHA domains in at least 20 otherwise unrelated proteins; most are from yeast but there are also some from mammals and bacteria. The typical FHA domain comprises approximately 55-75 amino acids and contains three highly conserved blocks separated by more divergent spacer regions. Only three positions seem to be invariant, including a histidine residue in the central part of the domain, which might be functionally important.
DNA polymerases are classified into four families on the basis of sequence similarities. DNA polymerase beta , from the eukaryotic 'X' family of DNA polymerases, is smaller (40 kDa) and simpler than the other polymerases. It operates in a stepwise rather than processive fashion, dissociating from the template-primer after the addition of each nucleotide. Here, we show by structure and sequence comparison that several remote relatives of DNA polymerase beta form a new superfamily of nucleotidyltransferases involved in diverse biological functions that range from DNA repair to regulation of biosynthetic pathways and antibiotic resistance.
Members of the highly conserved 14-3-3 family of proteins are found in a broad range of organisms, including higher eukaryotes, plants and invertebrates, and have been implicated in several signalling mechanisms in a variety of cell types. Since these proteins were first reviewed in this journal (in 1992), a near exponential increase in publications has suggested many additional biological roles for the 14-3-3 family. They have been reported in additional organisms including the nematode Caenorhabditis elegans and in halibut pituitary; a further mammalian isoform, called HME1 or stratifin has been found in human epithelial cells. Here, the author highlight some novel functions and isoforms of the 14-3-3 proteins, and in particular discuss the finding that has caused the most excitement recently: the interaction of specific 14-3-3 isoforms with Raf in the mitogen-activated protein kinase (MAPK) cascade.
The elongation factors EF-Tu and EF1 alpha have been investigated extensively in Escherichia coli and eukaryotic cells, respectively, for decades. These proteins have a high degree of structural and functional relatedness. Both belong to highly conserved protein families (>70% sequence identity in eukaryotes) that catalyse the GTP-dependent binding of aminoacyl-transfer RNA (aa-tRNA) to ribosomes, thereby regulating the fidelity and rate of polypeptide elongation during translation. In eukaryotes, EF1 alpha is the second most abundant protein after actin, constituting 1-2% of the total protein in normal growing cells. Large increases in mRNA levels for EF1 alpha are observed in rapidly proliferating cultured cells, embryos and rapidly proliferating cells in a variety of human tumors. These results indicate that the level of expression of EF1 alpha correlates with the rate of cell growth and proliferation. The mechanism behind this correlation is not obvious since EF1 alpha is normally present in vast molar excess to the other essential components of the translation machinery. The explanation for the abundance of EF1 alpha in eukaryotic cells and its importance in growth control may reside in unconventional properties recently discovered for this elongation factor.
Aeromonas sp. produce a lipolytic enzyme that also has the ability to carry out acyl transfer to alcohol acceptors in aqueous media. The fact that there is only one glycine in this sequence indicates that the structure of the active site is likely to differ from those with solved three-dimensional structures. There is evidence for the Escherichia coli thioesterase I and the Vibrio mimicus arylesterase that the first serine of this sequence is the nucleophile, as it is with the Aeromonas lipase. Proteins from E. coli, V. mimicus, Vibrio parahaemolyticus and Xenorhabdus luminescens are all known to have lipolytic activity.
Mass spectrometry is a venerable analytical tool that has been used for some time in biochemistry for the analysis of small molecules, such as steroids. More recently, physicists have solved the problems associated with Vaporizing and ionizing proteins and peptides, thereby allowing mass spectrometry to take on new roles in investigating protein sequences, structures and modifications.
G-protein-coupled receptor signaling and receptor tyrosine kinase (RTK) signaling are two mechanisms of transmembrane communication used by numerous extracellular agents and stimuli. The beta gamma-subunit complex of G proteins mediates many of the functions associated with G-protein-coupled receptor signaling and may even provide a means to link G proteins to RTK-initiated cascades. This connection may be mediated by the pleckstrin homology domain, a modular domain found in many signaling proteins that interact with G(beta gamma).
The signals required for forming 3'-ends of mRNAs from the yeast Saccharomyces cerevisiae differ from the corresponding signals of higher eukaryotes. Yeast signals consist of three elements: (1) the efficiency element, which enhances the efficiency of downstream positioning elements; (2) the positioning element, which positions the poly(A) site; and (3) the actual poly(A) site, These three elements are not only necessary, but also sufficient for mRNA 3'-end formation in yeast.
Post-transcriptional mechanisms contribute in many important ways to the overall control and regulation of gene expression, and in doing so employ a veritable army of proteins that bind a wide range of targets in messenger RNA (mRNA). The full range of these RNA-protein interactions is only just beginning to emerge, and much remains to be learned about the mechanisms underlying the rapidly increasing number of regulatory systems now being described.
A growing subset of small nucleolar RNAs (snoRNAs) contains long stretches of sequence complementarity to conserved sequences In mature ribosomal RNA (rRNA). This article reviews current knowledge about these complementarities and proposes that these antisense snoRNAs might function in pre-rRNA folding, base modification and ribosomal ribonucleoprotein assembly, in some cases acting as RNA chaperones.