Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of capdependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4␥, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.A ubiquitous feature of all eukaryotic cellular mRNAs is the presence at their 5Ј end of a cap structure, m7GpppX (where X is any nucleotide). The cap facilitates mRNA binding to ribosomes, a step which is rate limiting in translation (for reviews, see references 18 and 20). The cap is bound by the eukaryotic initiation factor 4F (eIF-4F) protein complex, which consists in mammals of three polypeptides: eIF-4E (a 25-kDa cap-binding protein), eIF-4␥ (also known as p220 in mammals), and eIF-4A (a 46-kDa RNA helicase). eIF-4F, in conjunction with eIF-4B, exhibits RNA helicase activity. Evidence suggests that this activity mediates mRNA unwinding in the vicinity of the cap structure in an ATP-dependent manner and facilitates binding of the 40S ribosomal subunit (26, 33). The 40S subunit is then thought to scan the mRNA in the 5Ј-to-3Ј direction until it encounters the initiator AUG codon. This series of steps constitutes the cap-dependent pathway of translation initiation (26,33).eIF-4E is the least abundant of all translation initiation factors, and its overexpression leads to deregulation of cell growth in HeLa cells (6) and to transformation of NIH 3T3 cells and CHO cells (5, 23) or rat primary fibroblasts, the latter in combination with v-myc or E1A (24). An attractive model for eIF-4E transforming activity is that overexpression of eIF-4E results in increased translation of mRNAs which are normally repressed, such as protooncogene mRNAs. eIF-4E activity is upregulated upon treatment of cells with growth factors, mitogens, and phorbol esters, which correlates with an increase in translatio...
Although estrogen receptors (ERs) recognize 15-bp palindromic estrogen response elements (EREs) with maximal affinity in vitro, few near-consensus sequences have been characterized in estrogen target genes. Here we report the design of a genome-wide screen for high-affinity EREs and the identification of approximately 70000 motifs in the human and mouse genomes. EREs are enriched in regions proximal to the transcriptional start sites, and approximately 1% of elements appear conserved in the flanking regions (-10 kb to +5 kb) of orthologous human and mouse genes. Conserved and nonconserved elements were also found, often in multiple occurrences, in more than 230 estrogen-stimulated human genes previously identified from expression studies. In genes containing known EREs, we also identified additional distal elements, sometimes with higher in vitro binding affinity and/or better conservation between the species considered. Chromatin immunoprecipitation experiments in breast cancer cell lines indicate that most novel elements present in responsive genes bind ERalpha in vivo, including some EREs located up to approximately 10 kb from transcriptional start sites. Our results demonstrate that near-consensus EREs occur frequently in both genomes and that whereas chromatin structure likely modulates access to binding sites, far upstream elements can be evolutionarily conserved and bind ERs in vivo.
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