RESUMEN
We wanted to discover whether the conformation of the mRNA leader sequence is involved in translational fidelity. For this purpose we constructed several mutants of Semliki Forest virus 26S mRNA and inserted AUG codons into the leader sequence. We then analyzed the results of in vitro and in vivo translation of these mRNAs, probed enzymatically the secondary structure and performed minimal energy folding of the transcripts. Our results indicate that the position of a hairpin in the leader sequence determines at which AUG codon downstream from that hairpin translation is initiated.
Asunto(s)
Codón , Regulación de la Expresión Génica , ARN Mensajero/análisis , Secuencia de Bases , ADN/análisis , Elementos Transponibles de ADN , Mutación , Conformación de Ácido Nucleico , Regiones Operadoras Genéticas , Biosíntesis de Proteínas , Virus de los Bosques Semliki/genética , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
We have investigated the minimal energy foldings of 38 mature mRNAs, including the globin family, the insulins, the growth hormones and interleukin-2, and have compared these foldings with those of fully and partly randomised sequences. The mRNAs differ from the random sequences in that they form a separate leader hairpin of 40-60 nucleotides, with the initiation codon typically located downstream of this hairpin, followed by a main fold in which a region flanking the initiation codon is basepaired with the trailer: resulting in a close proximity of the 5' and 3' end of the mRNA. The formation of this conformation depends not only--or primarily--on the structure of the leader, but on both the leader and trailer sequence and their interaction with the coding sequence. Thus if, as the frequent occurrence of this pattern suggests, the secondary structure of the leader regions plays a role in the initiation of translation, possibly accounting for the specificity of initiation and the different translational efficiencies of various mRNAs, we expect that these features may be influenced both by leader and trailer mutants.
Asunto(s)
Células/análisis , Células Eucariotas/análisis , Conformación de Ácido Nucleico , ARN Mensajero , Adenoviridae , Algoritmos , Secuencia de Bases , Globinas/genética , ARN ViralRESUMEN
Cap analysis of the late 26S Semliki Forest viral mRNA reveals that almost 30% of the caps possess both a methyl group at the N(7)-position and one at the N(2)-position. We have compared the degree of methylation of the caps of polysomal and non-polysomal 26S mRNA in order to check whether this feature is responsible for its translation late in infection. It was found that extra methyl groups on the caps cause a lower rate of initiation. Polysomal 26S mRNA contained less m2,7G- and m2,2,7G-caps than free 26S. The cap analog m2,2,7Gp was slightly less inhibitory than m7Gp in an in vitro translation system.