Single-strand DNA translation initiation step analyzed by Isothermal

abstract Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could bedirectly translated into protein. Some discrepancies in the results were reported about functional transla-tionofsingle-strandDNAbutallresultsconvergedonasimilarbehaviorofRNAandssDNAintheinitiationstep. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of inter-action between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the bindingwasnotaffectedbythenatureofthetemplatetestedandthedissociationconstantswereinthesamerangewhen ssDNA (K d = 3.62 ± 2.1 10 8 M) or the RNA corresponding sequence (K d = 2.7 ± 0.82 10 8 M)bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferenceswhich block the initiation complex formation. These results suggest that the limiting step in translationof ssDNA is the elongation process. 2009 Elsevier Inc. All rights reserved. IntroductionSince the discovery of the genetic code [1] and of messengerRNA (for review see [2]), the molecular biology dogma is anevident pathway for the expression from gene to protein. Never-theless, as early as 1964, DNA was used in vitro to study translationand the question still remains whether or not DNA could be di-rectly translated into protein. The first published works tried todetermine whether single-stranded DNA (ssDNA) might be ableto directly program the synthesis of proteins [3,4]. Single-strandedDNA and circular messenger DNA were then used in direct transla-tion systems for different purposes: to check the fidelity of thedirect translation [5], to demonstrate that ribosomes do not re-quire a free end on a messenger to initiate protein synthesis [6],to show that binding of fmet-tRNA and ssDNA to the ribosomecould occur without neomycin which is necessary for elongationwith these matrix [7], to test a synthetic ssDNA as ribosome bind-ing site [8], and finally, to make comparisons between RNA andssDNA in their ability to program ribosomes for initiation and ter-mination of translation [9]. In all papers cited above, all resultsconverge towards a similar behavior of RNA and ssDNA in the ini-tiation step, in spite of some discrepancy concerning the elonga-tion step of translation.The formation of the initiation complex involves many partnerswhich are largely described in the literature but the results aresometimes contradictory. For instance, the absence of initiationfactors, especially IF3, does not significantly affect the interactionof the 30S subunits of ribosome and mRNA [10], although its rolein promoting mRNA binding on ribosomes was demonstrated else-where [11]. On the other hand, the Shine–Dalgarno (SD) sequence[12], which is localized inside the ribosome binding site (RBS), isclearly involved in the interaction between mRNA and the 30Sribosome subunit. Therefore, this sequence is an unambiguous sig-nal to test the specificity of these interactions.In this biological process, the secondary or tertiary structures ofmRNA play a crucial role for the control of the translation initiation[13–15]. We choose to exclude this aspect because (i) RNA andssDNA do not shares the same secondary structures [16] (ii) thestructural organization of both RNA and ssDNA is difficult topredict and control in a biological context, even if fine structuralresults have pointed up the interactions between mRNA and 30Ssubunits [17,18].In this context, we tried to establish the thermodynamic valuesof this interaction in a global way, in order to determine and tocompare with a sensitive method the binding constants for these