Haloferax volcanii, a prokaryotic species that does not use the Shine Dalgarno mechanism for translation initiation at 5'-UTRs.
ABSTRACT: It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5'-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20-30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5'-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5'UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5'-UTRs. A genome analysis revealed that while the number of SD motifs in 5'-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons.
Project description: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.
Project description:The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.
Project description: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.
Project description:Three biological replicates of H. volcanii grown under optimal conditions to mid-exponential growth phase were used to determine the primary transcriptome and map 5â-ends of transcripts. In total, 4,749 potential transcriptional start sites (TSS) were detected. A position weight matrix was derived for promoter prediction, showing that 64% of the TSS were preceded by stringent or relaxed basal promoters. 1,851 TSS belonged to protein-coding genes, showing that less than half (46%) of the 4040 protein-coding genes are expressed under optimal growth conditions. 72% of all protein-coding transcripts were leaderless, underscoring that this is the default pathway for translation initiation in haloarchaea. The 5â-UTRs of transcripts with leaders had a widely varying length distribution without any optimum. 2,898 of the TSS belong to potential non-coding RNAs, representing an unexpectedly high fraction (61%) among all transcripts. 2792 of the non-coding TSS had not been described before and were thus novel (59% of all TSS). A large fraction of the potential novel non-coding transcripts are cis-antisense RNAs (1,244 aTSS). There was a strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs, suggesting that negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts correspond to internal transcripts overlapping with mRNAs (1,153 iTSS) and intergenic small RNA (sRNA) candidates (395 TSS). Three biological replicates were performed with slight differences in library preparation. In each case, part of the sample was treated with terminator 5'-phosphate-dependent exonuclease (+TEX), while part of the sample remained untreated (-TEX). Therefore, in total, six samples were analysed by high-throughput sequencing.
Project description:High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.
Project description:In recent years, synthetic riboswitches have become increasingly important to construct genetic circuits in all three domains of life. In bacteria, synthetic translational riboswitches are often employed that modulate gene expression by masking the Shine-Dalgarno (SD) sequence in the absence or presence of a cognate ligand. For (halo-)archaeal translation, a SD sequence is not strictly required. The application of synthetic riboswitches in haloarchaea is therefore limited so far, also because of the molar intracellular salt concentrations found in these microbes. In this study, we applied synthetic theophylline-dependent translational riboswitches in the archaeon <i>Haloferax volcanii</i>. The riboswitch variants A through E and E<sup>∗</sup> were chosen since they not only mask the SD sequence but also the AUG start codon by forming a secondary structure in the absence of the ligand theophylline. Upon addition of the ligand, the ribosomal binding site and start codon become accessible for translation initiation. Riboswitch E mediated a dose-dependent, up to threefold activation of the <i>bgaH</i> reporter gene expression. Raising the salt concentration of the culture media from 3 to 4 M NaCl resulted in a 12-fold increase in the switching capacity of riboswitch E, and switching activity increased up to 26-fold when the cultivating temperature was reduced from 45 to 30°C. To construct a genetic circuit, riboswitch E was applied to regulate the synthesis of the transcriptional activator GvpE allowing a dose-dependent activation of the <i>mgfp6</i> reporter gene under <i>P</i> <sub><i>pA</i></sub> promoter control.
Project description: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.In 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.
Project description:Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5'-UTRs and with very long 3'-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3'-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.
Project description:Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3' untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3' UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3' UTRs. The 3' UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3' UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3' UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3' UTRs of mRNAs may encode various products.
Project description:In prokaryotes, translation initiation is believed to occur through an interaction between the 3? tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5? untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5? UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.