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Leaderless translation is prevalent in haloarchaea, with many of these leaderless transcripts possessing short 5’-untranslated regions (UTRs) less than 10 nucleotides. Whereas, little is known about the function of this very short 5’-UTR. Our previous studies determined that just four nucleotides preceded the start codon of  hsp70  mRNA in  Natrinema  sp. J7, with residues -3A and +4G, relative to the A of the ATG start codon, acting as the preferred bases around the start codon of all known haloarchaeal  hsp70  genes. Here, we examined the effects of nucleotides flanking the start codon on gene expression. The results revealed that shortening and deletion of the short 5’-UTR enhanced transcript levels; however, it led to significant reductions in overall translational efficiency. AUG was efficiently used as start codons, in both the presence and absence of short 5’-UTRs. GUG also could initiate translation, even though it was so inefficient that it would not be detected without considerably elevated transcript. Nucleotide substitutions at position -4 to +6 were shown to affect gene expression by transcript and/or translational levels. Notably, -3A and A/U nucleotides at position +4~+6 were more optimal for gene expression. Nucleotide transversions of -3A to -3C and +4G to +4T with  hsp70  promoter from either  Haloferax volcanii  DS70 or  Halobacterium salinarum  NRC-1 showed the same effects on gene expression as that of  Natrinema  sp. J7. Taken together, our results suggest that the nucleotides flanking the start codon in  hsp70  mRNAs with very short 5’-UTRs play an important role in haloarchaeal gene expression.

Nucleotides Flanking the Start Codon in hsp70 mRNAs with Very Short 5’-UTRs Greatly Affect Gene Expression in Haloarchaea