RESUMEN
A wheat germ cell-free system was used to study details of ribosome shunting promoted by the cauliflower mosaic virus 35 S RNA leader. By testing a dicistronic construct with the leader placed between two coding regions, we confirmed that the 35 S RNA leader does not include an internal ribosome entry site of the type observed with picornavirus RNAs. A reporter gene fused to the leader was shown to be expressed by ribosomes that had followed the bypass route (shunted) and, with lower efficiency, by ribosomes that had scanned through the whole region. Stem section 1, the most stable of the three stem sections of the leader, was shown to be an important structural element for shunting. Mutations that abolished formation of this stem section drastically reduced reporter gene expression, whereas complementary mutations that restored stem section 1 also restored shunting. A micro-leader capable of shunting consisting of stem section 1 and flanking sequences could be defined. A small open reading frame preceding stem section 1 enhances shunting.
Asunto(s)
Caulimovirus/genética , Ribosomas/metabolismo , Composición de Base , Secuencia de Bases , Sistema Libre de Células , Cloranfenicol O-Acetiltransferasa/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genéticaRESUMEN
It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts.
Asunto(s)
Caulimovirus/genética , Plantas/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Badnavirus/genética , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , Oryza/virología , Plantas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero , ARN ViralRESUMEN
UV crosslinking assays were performed to characterize interactions between the leader of the cauliflower mosaic virus (CaMV) 35S RNA and cellular, cytoplasmic proteins. From turnip, a host for CaMV, three different proteins, p35, p49, and p100, interacted with multiple binding sites within the leader p35 binds to RNA nonspecifically and p49 binds with low specificity, whereas p100 interacts specifically with viral sequences. The expression of the proteins is not induced upon virus infection, as there is no difference in the protein pattern between healthy and infected cell extracts. In a cellular fractionation, p35 and p100 remain in the high-speed supernatant whereas p49 cosediments with polysomes. A possible involvement of these proteins in the translation of the CaMV 35S RNA is discussed.
Asunto(s)
Caulimovirus/metabolismo , Proteínas de Plantas/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Caulimovirus/genética , Datos de Secuencia Molecular , Mutación Puntual , Polirribosomas/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , VerdurasRESUMEN
The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (Lycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 bp long with a G + C content of 49.6%. Its sequence exhibits 94%-96% positional identity when it is colinearly aligned with the previously reported sequences of the 17-18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.