Small RNAs in haloarchaea: identification, differential expression and biological function.

To elucidate the role of small noncoding RNAs (sRNAs) in archaea we applied RNomics to identify sRNAs in the halophilic archaeon Haloferax volcanii. Using a size-selected cDNA library, 39 different previously uncharacterized sRNAs were identified ranging in size from 130 to 460 nucleotides. Twenty-one of these sRNAs are located in intergenic regions and 18 in antisense orientation. One of the intergenic sRNAs codes for a peptide. Only a minor fraction of sRNA genes were preceded by promoter elements (15 of 39), indicating that the majority might be generated by processing from larger precursors. Northern blot analyses of the intergenic sRNAs revealed differential expression for several sRNAs. Deletion mutants of two sRNAs were constructed, demonstrating that this approach is suitable to elucidate their biological function. Both mutant strains showed a defined phenotype: sRNA(30) gene deletion mutant was less resistant to higher temperatures and sRNA(63) gene deletion mutant resulted in a severe growth defect at low salt concentrations. Proteome analyses revealed clear differences between wildtype and deletion strains. These results represent the first reported examples of experimentally characterizing the function of sRNAs, excepting snoRNAs, in archaea. Taken together, we showed that haloarchaea encode sRNAs, some of which are differentially expressed and which have the potential to fulfil important biological functions in vivo.

Regulation of translation in haloarchaea: 5'- and 3'-UTRs are essential and have to functionally interact in vivo.

Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo.Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

A novel mechanism for translation initiation operates in haloarchaea.

Four different mechanisms for translation initiation are known, i.e. one prokaryotic mechanism involving a Shine-Dalgarno sequence, two eukaryotic mechanisms relying on ribosomal scanning or internal ribosomal entry sites, and one mechanism acting on leaderless transcripts. Recently it was reported that the majority of haloarchaeal transcripts is leaderless and that most leadered transcripts are devoid of a Shine-Dalgarno sequence, excluding the operation of a 'bacterial-like' initiation mechanism. Therefore, the current study concentrated on elucidating whether a 'eukaryotic-like' scanning mechanism might operate instead. GUG and UUG were efficiently used as start codons on leadered transcripts in vivo, in contrast to initiation on leaderless transcripts (and leadered eukaryotic transcripts). Deleted versions of the 5'-UTR initiated translation very inefficiently. Introduction of additional upstream AUGs did not influence the initiation efficiency at internal start codons. An additional in-frame AUG at the 5'-end led to the simultaneous usage of two start sites on the same message. A stable stem-loop structure at the 5'-end inhibited only initiation at the first AUG, but did not influence usage of the internal AUG. Taken together, operation of a scanning mechanism was excluded and the results indicate that a novel mechanism for translation initiation operates at least in haloarchaea.

Small RNAs of the halophilic archaeon Haloferax volcanii.

In recent years, sRNAs (small non-coding RNAs) have been found to be abundant in eukaryotes and bacteria and have been recognized as a novel class of gene expression regulators. In contrast, much less is known about sRNAs in archaea, except for snoRNAs (small nucleolar RNAs) that are involved in the modification of bases in stable RNAs. Therefore bioinformatic and experimental RNomics approaches were undertaken to search for the presence of sRNAs in the model archaeon Haloferax volcanii, resulting in more than 150 putative sRNA genes being identified. Northern blot analyses were used to study (differential) expression of sRNA genes. Several chromosomal deletion mutants of sRNA genes were generated and compared with the wild-type. It turned out that two sRNAs are essential for growth at low salt concentrations and high temperatures respectively, and one is involved in the regulation of carbon metabolism. Taken together, it could be shown that sRNAs are as abundant in H. volcanii as they are in well-studied bacterial species and that they fulfil important biological roles under specific conditions.

Experimental characterization of Cis-acting elements important for translation and transcription in halophilic archaea.

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.

Single chain Fab (scFab) fragment.

BACKGROUND: The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. RESULTS: Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabDeltaC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. CONCLUSION: A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.