Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

Abstract

Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5= untranslated region (5= UTR) of <10 nucleotides (nt) and leadered transcripts with a 5= UTR of >10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG 1 and AUG 2 ) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG 1 is more efficient than AUG 2 for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG 1 but did not affect initiation at AUG 2 , indicating that AUG 2 -initiated translation does not involve ribosome scanning from the 5= end of the transcript. Furthermore, the efficiency of AUG 2 -initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG 1 and AUG 2 contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea. IMPORTANCEIn eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

Halolysin SptA, a Serine Protease, Contributes to Growth-Phase Transition of Haloarchaeon Natrinema sp. J7-2, and Its Expression Involves Cooperative Action of Multiple Cis-Regulatory Elements

“…The core promoter (-43 to -1) and transcript (+1 to +1925) of sptA were previously determined ( Figure 1A ) ( Tang et al, 2016 ). In order to investigate the function of SptA, the mutant Δ sptA1 was created with deletion of the core promoter and entire coding region (-43 to +1655) of sptA in Natrinema sp.…”

“…J7-2 genomic DNA template was combined with different forward primers (up215-F to up43-F) and a common reverse primer (up-bgaH-R) to amplify sptA 5′-flanking region of different lengths (megaprimers). Subsequently, bgaH was amplified from plasmid pST1 ( Tang et al, 2016 ) using the megaprimer and the primer bgaH-HindIII-R. The PCR products were individually cloned into the BamHI-HindIII restriction site of pYC-SHSmcs to generate the plasmids pD215b to pD43b.…”

“…Genomic DNA removal and reverse transcription were conducted using a PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time; Takara) according to the manufacturer’s instructions. The qRT-PCR was carried out in a 20 μl reaction mixture as described previously ( Tang et al, 2016 ). The sptA transcript levels were determined using the primer pair SptAQRT-F/SptAQRT-R. As an internal control, the 16S rRNA transcript levels were determined with the primer pair 16SrRNAQRT-F/16SrRNAQRT-R.…”

“…The enzymatic properties, autocatalytic activation, and Tat-dependent secretion mechanisms of SptA have been characterized ( Shi et al, 2006 ; Xu et al, 2011 ; Du et al, 2015 ). Recently, the core promoter and transcript of the sptA gene were experimentally determined ( Tang et al, 2016 ). The results showed that the sptA core promoter contains some highly conserved, cis -acting elements of haloarchaeal promoters, including a transcription factor B (TFB) recognition element (BRE) motif, a TATA box recognized by the TATA-binding protein (TBP), and a WW motif that possibly interacts with TFB and RNA polymerase (RNAP) ( Brenneis et al, 2007 ).…”

Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing

“…An artificial mini-CRISPR, comprising two repeats separated by a spacer matching a PAM (TTC)-preceded sequence (protospacer) within crtB , as well as a donor DNA fragment were inserted into pSHS, yielding the self-targeting plasmid pKC; the mini-CRISPR is under the control of the sptA promoter ( 48 ) ( Fig. 3A ).…”