Ribosomal selection of mRNAs with degenerate initiation triplets

Published online 27 May 2017 Nucleic Acids Research, 2017, Vol. 45, No. 12 7309–7325 doi: 10.1093/nar/gkx472 Ribosomal selection of mRNAs with degenerate initiation triplets He Chengguang1,2,† , Paola Sabatini2,† , Letizia Brandi2 , Anna M. Giuliodori2,* , Cynthia L. Pon2 and Claudio O. Gualerzi2,* 1 College of Life Sciences, Engineering Research Centre of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, Jilin, China and 2 Laboratory of Genetics, University of Camerino 62032 Camerino, Italy Received March 18, 2017; Revised April 20, 2017; Editorial Decision May 03, 2017; Accepted May 12, 2017 ABSTRACT To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40◦ C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing nonAUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present. INTRODUCTION It has been known since the late 60’s that translation initiation begins with the 30S ribosomal subunit which forms a complex with mRNA and initiator tRNA (e.g. ref. 1,2). The possibility of initiating protein synthesis with undisso- ciated 70S ribosomes is restricted to the cases in which the template is a polynucleotide such as polyuridylic acid or a leaderless mRNA (3) or when the subunits are artificially prevented from dissociating as a result of crosslinking (4). Formation of a 30S translation initiation complex (30SIC) represents a key step within the whole process of protein synthesis. The small ribosomal subunit bearing one copy each of the three initiation factors IF1, IF2 and IF3 binds in stochastic order an mRNA and an initiator tRNA (fMet-tRNA) molecule (for reviews see 5–7). Together, these ribosomal ligands are assembled in a 30S preinitiation complex (30S preIC) in which codon-anticodon interaction has not yet occurred or is incomplete (8–10). A first-order transition which likely involves a structural modification of the highly conserved GGAA tetraloop of h45 (G1516–A1519) and the conversion of the h44/h45/h24a interface from an ‘open’ to a ‘closed’ conformation accompanies codon–anticodon interaction in the P-site and marks the transformation of the 30S preIC into a 30SIC (8,10). A 30SIC containing canonical ligands and endowed with a canonical structure is amenable for docking by the 50S subunit and enters the subsequent stages of the translation initiation pathway whose epilogue consists in the formation of a 70S initiation complex (70SIC) and of the first peptide bond yielding the initiation dipeptide (11). The 30S preIC → 30SIC transition is under the kinetic control of the initiation factors and represents the first checkpoint of translational fidelity (8,11,12). In fact, both on- and off-rates of the transition are increased by IF3 but the off-rates are affected to different extents, depending upon the nature of the 30S ligands and upon the structural properties of the resulting complex. In this way non-canonical complexes are dissociated and discriminated against. Indeed, at least four different types of 30S complexes regarded as non-canonical are rejected by IF3 (5,13,14) with the assistance of IF1 (11,15) and have little or no chance to enter the later stages of translation initiation. In particular, the nature of the initiation triplet * To whom correspondence should be addressed. Tel: +39 0737 403240; Fax: +39 0737 403290; Email: Correspondence may also be addressed to Anna M. Giuliodori. Tel: +39 0737 403 251; Fax: +39 0737 403 290; Email: † These authors contributed equally to the paper as first authors.  C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 7310 Nucleic Acids Research, 2017, Vol. 45, No. 12 present within the translational initiation region (TIR) of the mRNA represents one of the discriminants targeted by IF3; both in vitro (13,14,16) and in vivo (17,18) analyses have shown that only three start codons (AUG, GUG and UUG) are accepted by IF3 as ‘canonical’ whereas all the others, regarded as ‘non-canonical’, are rejected by the factor, albeit to different levels during 30SIC formation. Thus, the mRNAs using non-canonical start codons are subject to translation repression by this factor and occasionally expressed at low levels. A typical example is represented by the infC gene which uses either AUU or AUC in all bacterial species and is auto-regulated by its own gene product IF3 (19–22). Genomic analysis of over 600 bacterial species revealed that the canonical triplets AUG (80.1%), GUG (11.6%) and UUG (7.8%) are the most frequent start codons (23) whereas non-canonical degenerate initiation triplets are extremely rare. Nevertheless, other triplets like AUA, AUC and AUU are also found; although the latter is present in only two Escherichia coli genes (infC and pcnB) (24,25), AUU and AUC are found quite frequently in other species such as Mycoplasma gallisepticum (23), the causative agent of avian chronic respiratory disease. Non-canonical triplets such as CUG, AUU, AUC and AUA are found in low percentage (between 0.004 and 0.024%) among all the start codons annotated from 79 bacterial genome and plasmid sequences (26). However, these figures might well represent an underestimation of the occurrence of these initiation triplets because initiation codon identification in bacteria is far from being precise, mainly in light of the generally very low-level of expression of genes containing these uncommon start codons. It is noteworthy in this connec (...truncated)