S1, a multi-domain ribosomal protein associated with the 30S subunit, is essential for translation initiation. S1 binds with high affinity to single-stranded mRNA containing AU-rich patches upstream of the start codon. It was previously reported that domains 1-3 of S1 protein play a role in the docking and unfolding of structured mRNAs to the ribosome. Moreover, S1-deficient 30S subunits are still able to bind to low structured mRNAs. However, mRNAs containing AU-rich patches in the early base positions after start codon enhance protein synthesis and mRNA binding to the ribosome, which suggests that S1 is also able to interact with these AU-rich regions. To evaluate the essentiality of S1 domains in the binding to low structured mRNAs containing A/U/G nucleotides after the start codon as well as their role in translation and cell viability, S1 protein deletion variants were generated. We show that S1 domain 3 is necessary to discriminate these mRNAs according to the nucleotide nature since its absence abrogated S1 binding to A/U-rich mRNAs and allowed binding to G-rich mRNAs. Interestingly, domains 2-3 were required for the binding of mRNAs containing A/U-rich sequences after the start codon to 30S, in vitro translation, and cell viability.