Abstract
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.
Highlights
Archaea possess many features distinct from bacteria and eukaryotes
The archaeal basal transcription apparatus consists of a single eukaryotic RNA polymerase (RNAP) II-like transcriptase and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB)
J7, the 5’-untranslated regions (UTRs) of hsp70, determined by RLM-RACE PCR (RNA ligase-mediated rapid amplification of cDNA ends by PCR), was just 4 nucleotides in Hbt. salinarum NRC-1 and Hfx. volcanii DS70, which suggested that haloarchaeal hsp70 mRNAs were likely to be with very short 5’-UTR
Summary
Archaea possess many features distinct from bacteria and eukaryotes. The archaea are prokaryotic organisms closely related to bacteria in morphology and metabolism. Many aspects of their informational processes are more related to that of PLOS ONE | DOI:10.1371/journal.pone.0138473. Nucleotides Flanking the Start Codon in hsp mRNAs their eukaryotic homologues [1, 2]. The archaeal basal transcription apparatus consists of a single eukaryotic RNA polymerase (RNAP) II-like transcriptase and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB). TBP and TFB are homologues of the eukaryotic TBP and basal transcription factor TFIIB. The archaeal promoter architecture containing the consensus sequence TATA box closely resembles that of the eukaryotic RNAP II promoters in terms of nucleotide sequence, location and function [3]
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