Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

Rikard Dryselius¹^,2 , Abbas Nikravesh¹ , Agne Kulyté¹^,3 , Shan Goh¹ and Liam Good¹

¹Department of Cell and Molecular Biology, Programme for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, 171 77, Stockholm, Sweden

²Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita City, Osaka, 565-0871, Japan

³Life Sciences, Södertörns University College, Alfred Nobels allé 3, 14152 Huddinge, Sweden

author email corresponding author email

BMC Microbiology 2006, 6:97doi:10.1186/1471-2180-6-97


Published:	21 November 2006

Abstract

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.

Conclusion

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.