RESUMO
The internal light-regulatory element (iLRE) of ferredoxin (Fed-1) mRNA, comprising the 5' leader and at least the first 13 codons of the open reading frame, controls transcript abundance after illumination of the plant in a translation-dependent manner. We have characterized the RNA binding activities associated with the Fed-1 iLRE and have identified one activity as the heat shock protein HSP101, a protein shown to bind the 5' leader of tobacco mosaic virus. HSP101 was sufficient and necessary to mediate a high level of translational activity from a Fed-1 iLRE-containing mRNA in yeast. Moreover, the Fed-1 iLRE substantially enhanced translation of reporter mRNAs in plant protoplasts expressing HSP101. Expression of HSP101 was subject to developmental regulation in leaves in that expression was highest in young leaves. These data suggest that Fed-1 mRNA may use the HSP101 regulatory mechanism as a means of ensuring a high level of translation required for the light-mediated regulation of Fed-1 mRNA stability.
Assuntos
Ferredoxinas/genética , Proteínas de Choque Térmico/metabolismo , Biossíntese de Proteínas , Sítios de Ligação , Ferredoxinas/metabolismo , Luz , Plantas/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismoRESUMO
The 5' leader (Omega) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102-kD protein, identified previously as a specific Omega RNA-binding protein, revealed homology to the HSP101/HSP104/ClpB family of heat shock proteins and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 50-fold increase in the translation of Omega-luc, but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas Omega failed to enhance translation in the absence of HSP101. Therefore, HSP101 and Omega comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factor (eIF) 3 and specifically one (TIF4632) of the two eIF4G proteins were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells or in cells subject to nutrient limitation, but its thermotolerance function remained unaffected. This is the first identification of a protein required for specific translational enhancement of capped mRNAs, the first report of a translational regulatory function for any heat-shock protein, and the first functional distinction between the two eIF4G proteins present in eukaryotes.
Assuntos
Proteínas de Choque Térmico/fisiologia , Proteínas de Plantas/fisiologia , Biossíntese de Proteínas , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Respiração Celular/genética , Fator de Iniciação 3 em Eucariotos , Fator de Iniciação Eucariótico 4G , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Dados de Sequência Molecular , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Tóxicas , Poli A/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Triticum/genética , Triticum/metabolismoRESUMO
PURPOSE: Recent information indicates that large, sustained wall shear stress gradients are a dominant hemodynamic parameter associated with the location and severity of atherosclerosis and myointimal hyperplasia. This study computes the spatial values of wall shear stresses and their gradients for three carotid artery bifurcation geometries. METHODS: A computational fluid dynamics program was used to solve the transient two-dimensional partial differential equations that describe fluid flow. Blood was treated as both a Newtonian and a non-Newtonian incompressible fluid. Solutions for the velocities, wall shear stresses, and wall shear-stress gradients were obtained for three carotid bifurcation geometries: a normal carotid bifurcation (similar to a primarily reconstructed carotid endarterectomy), a patch-reconstructed carotid endarterectomy, and a gradually tapered, low-angle carotid bifurcation (no carotid bulb). RESULTS: Computed velocity profiles closely match published experimental ones. Disturbed flow velocities are largest in the bulb segment of the normal carotid bifurcation. Peak and minimum wall shear stresses and peak shear stress gradients occurred in the lateral internal carotid artery wall. These were binodal in the normal or primarily reconstructed carotid artery, localized at the distal end of the patch-reconstructed carotid bifurcation, and minimal in the smooth, tapered carotid bifurcation. Wall shear stresses and their gradients were slightly higher for non-Newtonian than Newtonian fluids in the normal carotid artery but were similar in the other two geometric configurations. CONCLUSION: These results indicate that flow disturbances in general and wall shear stress gradients in particular are markedly reduced in carotid artery bifurcations that are smooth and gradually tapered and do not have a bulb. Abrupt geometric wall changes such as those occurring in the normal carotid bulb and at the distal end of a patch-reconstruction after carotid endarterectomy are harbingers of disturbed flow and high wall shear stress gradients. These results suggest that carotid endarterectomy reconstruction geometry characterized by a gradually tapered internal carotid artery may minimize the hemodynamically induced component of early myointimal hyperplasia and thrombosis and late atherosclerotic restenosis.
