BMB reports 568 BMB reports http://bmbreports.org *Corresponding author. Tel: 39-051-2093671; Fax: 39-051-2093673; E-mail: hochko@ms.fci.unibo.it Received 1 December 2007, Accepted 4 January 2008 Keywords: Antisense RNAs, E. coli, lacZ, Shine-Dalgarno sequence, Transcription Artificial antisense RNAs silence lacZ in E. coli by decreasing target mRNA concentration Stefan Alessandra 1,2 , Tonelli Alessandro 1 , Schwarz Flavio 1 & Hochkoeppler Alejandro 1,2, * 1 Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, 40136 Bologna, 2 CSGI, University of Florence, Italy Antisense RNA molecules are powerful tools for controlling the expression of specific genes but their use in prokaryotes has been limited by their unpredictable antisense effectiveness. Moreover, appreciation of the molecular mechanisms associated with si- lencing in bacteria is still restricted. Here we report our attempts to define an effective antisense strategy in E. coli, and to dissect the observed silencing process. Antisense constructs comple- mentary to different regions of lacZ were investigated, and silenc- ing was observed exclusively upon expression of antisense RNA hybridising the 5'UTR of lac messenger. The level of lacZ mRNA was reduced upon expression of this antisense construct, and the silencing competence was found to be closely associated with its stability. These observations may help in the design of antisense molecules directed against prokaryotic genes. [BMB reports 2008; 41(8): 568-574] INTRODUCTION Conventional bacterial genetics examines the essential genes by selecting temperature-sensitive alleles or by promoter replace- ment. The first approach is useful for selecting inter-genic sup- pressors, while the second strategy provides strains capable of expressing the gene of interest at different levels. Since the con- struction of this type of strain is hampered by the low recombi- nation proficiency of several prokaryotes, improved allele re- placement methodologies, such as the use of optimised suicide vectors (1) or recombineering (2), have been proposed. However, promoter replacement cannot be used to obtain the conditional expression of a gene among the different cistrons belonging to the same operon. On the contrary, a trans-acting element able to tune gene expression can target the individual cistrons in- dependently of their location in a particular bacterial operon. The efficacy of such a strategy becomes clear in biochemical and metabolic studies, which would greatly benefit the se- lective tools that can tune, in vivo, the copy number of specific proteins. The conditional repression of prokaryotic genes is therefore a major challenge for genetic, biochemical and in- dustrial purposes. The use of antisense molecules to conditionally repress pro- karyotic gene expression has been pursued for a number of years. Two main strategies are currently being challenged: i) the use of antisense peptide-nucleic acids (PNAs); ii) the condi- tional expression, in vivo, of antisense RNAs (asRNAs). Anti- sense PNAs are generally considered efficient silencers (3), even though they are not readily delivered to the cells. This makes it difficult to determine a dose/effect relationship. The in vivo expression of asRNAs may be a promising approach for the conditional silencing of target genes and the identification of essential genes (4). However, their general use is still ham- pered by the lack of a robust design strategy. Moreover, while knowledge of the mechanism of gene repression exerted by natural antisense RNAs has increased steadily over recent years (5), there are only two reports (6, 7) showing partial data on the molecular events associated with silencing by artificial asRNAs in prokaryotes. It should also be noted that the occur- rence in prokaryotes of genes coding for the functional ana- logues of the enzymes engaged in eukaryotic RNA interference was quite recently hypothesized (8), but there is no experi- mental evidence supporting this possibility. The following points need to be addressed in order to de- vise an effective asRNA: i) the region of a gene sequence that represents the most appropriate target of the antisense tran- script; ii) the length of asRNA that maximizes its performance; and iii) the factors affecting antisense stability in vivo and, hence, silencing. While conflicting data concerning the best target region and size of asRNAs have been reported (9, 10), there is almost no information available on the relationship between asRNA sta- bility and silencing competence. Recently, it was reported that the presence of paired termini at the 5' and 3' sides of the anti- sense transcripts results in improved stability in vivo (7). Al- though these antisenses proved to be efficient silencers, the presence of inverted repeats in these constructs might lower their genetic stability. Our ongoing search for asRNAs competent in silencing dnaQ (coding for the proofreading subunit of DNA polymer- ase III) in E. coli led to the identification of the pBAD-AR1 tran-