[lambda N gene expression regulated by the leader sequence].
It was reported recently that changing the TIR (translational initiation region) of lambda N gene resulted in the increasing expression of lambda N gene and it was regulated at translational level. According to the alignment, the leader sequence of lambda N gene had three parts: a code region for ORF lambda N, the upstream sequence of ORF lambda N and 17 bp of spacer between ORF lambda N and downstream of lambda N gene. ORF lambda N is an open reading frame located at upstream of lambda N gene coding a peptide of 19 amino acids. To study the mechanism of regulation of lambda N gene expression, three serials of plasmids with mutant leader region of lambda N gene were constructed. (1) pMC1403-XT, in which the start codon or the partial code of ORF lambda N was connected with lacZ to obtain the ORF lambda N-lacZ fusion gene and in which the ORF lambda N-lacZ expression was under the control of a strong trp/lac promoter; (2) The ORF lambda N mutants in which the termination codon TAA was introduced into ORF lambda N at different positions by site-directed mutagenesis to preterminate the ORF lambda N on plasmid pMC1403N; (3) Mutants in which a deletion was located at upstream ORF lambda N in the ORF lambda N mutants above. The results obtained from determination of the beta-galactosidase activity in the transformants harboring the different plasmids showed that the ORF lambda N-lacZ expression was suppressed by the ORF lambda N code region and the lambda N expression was increased in both the second and third serials of mutants. At the same time the results from RNA-DNA dot hybridization showed that the lambda N gene expression was regulated at translational level. Therefore it was predicted that the reason of relatively low expression for lambda N gene in pMC1403N was due to the low efficiency of ORF lambda N translation. There are two ways to increase the expression of lambda N gene. One is to preterminate the translation of ORF lambda N at a suitable position to decrease the ORF lambda N effects on downstream lambda N gene translation; the other is to change the upstream sequence of ORF lambda N to improve its translation efficiency.
Hu
Q R
QR
Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China. grhu@genetics.ac.cn
Han
B M
BM
Li
M Y
MY
Guo
L H
LH
Zhao
R L
RL
Weng
M L
ML
D020121:5' Untranslated Regions / Q000502:physiology*; D010582:Bacteriophage lambda / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D016366:Open Reading Frames; D014176:Protein Biosynthesis
D020121:5' Untranslated Regions