Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea

Abstract

The basal transcription apparatus of archaea is well characterized. However, much less is known about the mechanisms of transcription termination and translation initation. Recently, experimental determination of the 5′-ends of ten transcripts from Pyrobaculum aerophilum revealed that these are devoid of a 5′-UTR. Bioinformatic analysis indicated that many transcripts of other archaeal species might also be leaderless. The 5′-ends and 3′-ends of 40 transcripts of two haloarchaeal species, Halobacterium salinarum and Haloferax volcanii, have been determined. They were used to characterize the lengths of 5′-UTRs and 3′-UTRs and to deduce consensus sequence-elements for transcription and translation. The experimental approach was complemented with a bioinformatics analysis of the H. salinarum genome sequence. Furthermore, the influence of selected 5′-UTRs and 3′-UTRs on transcript stability and translational efficiency in vivo was characterized using a newly established reporter gene system, gene fusions, and real-time PCR. Consensus sequences for basal promoter elements could be refined and a novel element was discovered. A consensus motif probably important for transcriptional termination was established. All 40 haloarchaeal transcripts analyzed had a 3′-UTR (average size 57 nt), and their 3′-ends were not posttranscriptionally modified. Experimental data and genome analyses revealed that the majority of haloarchaeal transcripts are leaderless, indicating that this is the predominant mode for translation initiation in haloarchaea. Surprisingly, the 5′-UTRs of most leadered transcripts did not contain a Shine-Dalgarno (SD) sequence. A genome analysis indicated that less than 10% of all genes are preceded by a SD sequence and even most proximal genes in operons lack a SD sequence. Seven different leadered transcripts devoid of a SD sequence were efficiently translated in vivo, including artificial 5′-UTRs of random sequences. Thus, an interaction of the 5′-UTRs of these leadered transcripts with the 16S rRNA could be excluded. Taken together, either a scanning mechanism similar to the mechanism of translation initiation operating in eukaryotes or a novel mechanism must operate on most leadered haloarchaeal transcripts.

Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

“…qRT-PCR was performed on total RNA preparations as previously described (Sherwood et al, 2009) using the iQ SYBR Green Supermix and iCycler MyiQ real-time PCR detection system (Bio-Rad) with primers specific for smrs-gfp and ribL10 (ribosomal protein). The ribL10 primers (RibL-RT_f and RibL-RT_r) were as reported elsewhere and served as a normalization control (Brenneis et al, 2007). The smrs-gfp primers were Fw, 59-ACGACTTCTTCAAGAGCG-39, and Rev, 59-CTGCCGTGATGT-ATACGT-39.…”

“…Primer pairs were designed to detect the levels of smrs-gfp-specific transcript in total RNA preparations. In addition, the ribL10-specific transcript levels (encoding a ribosomal protein) were determined and served as an unregulated transcript control (Brenneis et al, 2007). The qRT-PCR analysis revealed no dramatic difference in the levels of smrs-gfp-specific transcript among the various strains (i.e.…”

Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability

“…Long 5Ј UTRs in methanoarchaea were completely unexpected, because previous genomic and experimental analyses had predicted archaeal mRNAs to either be leaderless or possess rather short 5Ј UTRs (27). In addition, a recent systematic screen showed that the majority of haloarchaeal transcripts might be leaderless (28), and a very recent RNA-seq analysis (52) demonstrated that leaderless mRNAs are the rule in S. solfataricus P2. In contrast, the high number of long 5Ј UTRs argue for extensive posttranscriptional regulation at 5Ј UTRs in methanoarchaea (e.g., at the level of transcript stability) by regulatory proteins or RNAs, or through riboswitches.…”

ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis

“…S2 in the supplemental material). This finding was not surprising, as leaderless transcripts are common to haloarchaea (20,21). An initial analysis was performed in wt cells to assess whether thiamine and/or its metabolic precursors could influence reporter gene expression.…”