RESUMO
It is thought that Shine-Dalgarno-like sequences, which exhibit complementarity to the nucleotide sequences at the 3'-end of 18S rRNA, are not present in eukaryotic mRNAs. However, complementary sequences consisting of more than 5 nucleotides to the 3'-end of 18S rRNA, i.e., a CR sequence, are present at -17 to -32 upstream from the initiation codon AUG in 18 mRNAs involved in protein synthesis except eEF1A mRNA. Thus, effects of the CR sequence in mRNAs and polyamines on protein synthesis were examined using control and polyamine-reduced FM3A and NIH3T3 cells. Polyamines did not stimulate protein synthesis encoded by 18 mRNAs possessing a normal CR sequence. When the CR sequence was deleted, protein synthetic activities decreased to less than 70% of intact mRNAs. In eEF1A mRNA, the CR sequence was located at -33 to -39 upstream from the initiation codon AUG, and polyamines stimulated eEF1A synthesis about threefold. When the CR sequence was shifted to -22 to -28 upstream from the AUG, eEF1A synthesis increased in polyamine-reduced cells and the degree of polyamine stimulation decreased greatly. The results indicate that the CR sequence exists in many eukaryotic mRNAs, and the location of a CR sequence in mRNAs influences polyamine stimulation of protein synthesis.
Assuntos
Códon de Iniciação/metabolismo , Fator 1 de Elongação de Peptídeos/biossíntese , Poliaminas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 18S/metabolismo , Animais , Linhagem Celular Tumoral , Códon de Iniciação/genética , Camundongos , Fator 1 de Elongação de Peptídeos/genética , Biossíntese de Proteínas/fisiologia , RNA Ribossômico 18S/genéticaRESUMO
We have reported that polyamines increase cell viability at the stationary phase of cell growth through translational stimulation of ribosome modulation factor, and SpoT and RpoZ proteins involved in the synthesis and function of ppGpp in Escherichia coli. Since biofilm formation is also involved in cell viability, we looked for proteins involved in biofilm formation and cell viability whose synthesis is stimulated by polyamines at the level of translation. It was found that the synthesis of response regulators UvrY and CpxR in the two-component signal transducing systems and ribosome recycling factor (RRF) was increased by polyamines at the level of translation. Polyamine stimulation of the synthesis of UvrY and RRF was dependent on the existence of the inefficient initiation codons UUG and GUG in uvrY and frr mRNA, respectively; and polyamine stimulation of CpxR synthesis was dependent on the existence of an unusual location of a Shine-Dalgarno (SD) sequence in cpxR mRNA. Biofilm formation and cell viability in the absence of polyamines was increased by transformation of modified uvrY and cpxR genes, and cell viability by modified frr gene whose translation occurs effectively without polyamines. The results indicate that polyamines are necessary for both biofilm formation and cell viability.
