DOI: 10.1002/ijch.201000006 Role of the Initiation Factors in mRNA Start Site Selection and fMet-tRNA Recruitment by Bacterial Ribosomes Claudio O. Gualerzi,* [a] Attilio Fabbretti, [a] Letizia Brandi, [a] Pohl Milon, [a] and Cynthia L. Pon [a] 1. Introduction Initiation is the first step of protein synthesis, in which the small ribosomal subunit, with the help of three initia- tion factors (IFs) IF1, IF2, and IF3, recruits both the translation initiation region (TIR) of the mRNAs and ini- tiator tRNA (fMet-tRNA) to form a “30S initiation com- plex” (30SIC); the latter is joined by the large (50S) ribo- somal subunit to yield a “70S initiation complex” (70SIC). Upon formation of the first peptide bond with the incoming aminoacyl-tRNA (initiation dipeptide), the ribosome proceeds through the elongation and termina- tion steps of protein synthesis, the latter including the so- called “ribosome recycling” step. [1] Since the structural as- pects of the IFs have been reviewed recently, [2] here we summarize mainly the functional aspects of translation in- itiation. 2. Mechanistics of Translation Initiation Translation initiation is a multistep process in which, unlike thermodynamic aspects which are of marginal rele- vance (see below), kinetic aspects play a major role in controlling both efficiency and fidelity. This premise is now widely accepted, but it was short of being heretical when, in the mid-1970 s, the mechanistic aspects of trans- lation initiation started to be investigated and unravelled by kinetic analysis. [3] In the midst of a fierce dispute be- tween different schools of thought as to whether mRNA or fMet-tRNA binds first to the 30S subunit, these early studies showed that (i) the order of binding is stochastic and not obligatory, which led to the main conclusions that (ii) a first order isomerisation of a “30S pre-initiation complex”, entailing the P-site decoding of the initiation triplet by the fMet-tRNA, is rate-limiting in 30SIC forma- tion. Furthermore, while it was taken for granted that the IFs affect the thermodynamic affinity of the ribosome for its ligands (e. g., IF3 was believed to bind the mRNA to the 30S subunit, and “different forms”—in reality, differ- ent degradation products of this facto—were believed to bind different “classes” of mRNAs) it was demonstrated that the three IFs are kinetic effectors of the isomerisa- tion (steps 7,8 in Figure 1 A), [3b, c] which they influence in different ways depending upon the nature of the 30S li- gands. [4] Another dogma at that time was that, by analogy with elongation factor EF-Tu and eukaryotic initiation factor eIF2, also bacterial IF2 functions as a tRNA carri- er. Fast kinetic analyses using fluorescent N-AcPhe- tRNA as an fMet-tRNA ersatz led to the opposite con- clusion, namely, that IF2 encounters the initiator tRNA on the ribosome and not in solution. [3e] An identical con- clusion has been reached in a much more recent study in Abstract : Translation initiation is a complex, multi-step pro- cess of fundamental importance in all kingdoms of life, during which the start site of the genetic message transmit- ted in the form of an RNA molecule (mRNA) is selected, and the level of translation determined. Being the slowest step of protein synthesis, initiation is the phase most often subject to regulation. Here we review, in a historical per- spective and focusing mainly on results from our laboratory, the development of our perception of the mechanisms by which the most relevant steps of this pathway occur in bac- teria. In particular, we describe: (a) the mechanistics and ki- netics of translation initiation; (b) properties of mRNAs with and without Shine–Dalgarno sequence relevant for ini- tiation site selection and translational efficiency; c) riboso- mal binding and dissociation of the initiation factors, forma- tion and properties of translation initiation intermediates ; (d) the mechanisms by which translation initiation fidelity is ensured. Finally, we provide a short survey of the known translation initiation inhibitors. Keywords: protein synthesis · translation initiation factors · mRNA properties · translation initiation fidelity [a] C. O. Gualerzi, A. Fabbretti, L. Brandi, P. Milon, C. L. Pon Laboratory of Genetics, Department of Biosciences & Biotech- nology, University of Camerino, 62032 Camerino, Italy phone: + 39 (0)73403240 fax: + 39 (0)73403290 e-mail: claudio.gualerzi@unicam.it 80  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Isr. J. Chem. 2010, 50, 80 – 94 Review C.O. Gualerzi et al.