Variable coordination of cotranscribed genes in Escherichia coli following antisense repression.
BACKGROUND
A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target.


RESULTS
To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment.


CONCLUSIONS
The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.
Dryselius
Rikard
R
Department of Cell and Molecular Biology, Programme for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, 171 77, Stockholm, Sweden. rikard@biken.osaka-u.ac.jp
Nikravesh
Abbas
A
Kulyté
Agne
A
Goh
Shan
S
Good
Liam
L
D018387:Codon, Initiator; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis; D007763:Lac Operon / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D020135:Peptide Nucleic Acids / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D018387:Codon, Initiator; D008164:Luminescent Proteins; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins