Crystal structure of glycosyltrehalose synthase fromSulfolobus shibataeDSM5389

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeonSulfolobus shibataeDSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α)8-barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

Biochemical and Structural Insights into RNA Binding by Ssh10b, a Member of the Highly Conserved Sac10b Protein Family in Archaea

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.

Archaeal transcription: making up for lost time

In recent years, emerging structural information on the aRNAP (archaeal RNA polymerase) apparatus has shown its strong evolutionary relationship with the eukaryotic counterpart, RNA Pol (polymerase) II. A novel atomic model of SshRNAP (Sulfolobus shibatae RNAP) in complex with dsDNA (double-stranded DNA) constitutes a new piece of information helping the understanding of the mechanisms for DNA stabilization at the position downstream of the catalytic site during transcription. In Archaea, in contrast with Eukarya, downstream DNA stabilization is universally mediated by the jaw domain and, in some species, by the additional presence of the Rpo13 subunit. Biochemical and biophysical data, combined with X-ray structures of apo- and DNA-bound aRNAP, have demonstrated the capability of the Rpo13 C-terminus to bind in a sequence-independent manner to downstream DNA. In the present review, we discuss the recent findings on the aRNAP and focus on the mechanisms by which the RNAP stabilizes the bound DNA during transcription.

Archaeal RNA polymerase: the influence of the protruding stalk in crystal packing and preliminary biophysical analysis of the Rpo13 subunit

We review recent results on the complete structure of the archaeal RNAP (RNA polymerase) enzyme of Sulfolobus shibatae. We compare the three crystal forms in which this RNAP packs (space groups P212121, P21212 and P21) and provide a preliminary biophysical characterization of the newly identified 13-subunit Rpo13. The availability of different crystal forms for this RNAP allows the analysis of the packing degeneracy and the intermolecular interactions that determine this degeneracy. We observe the pivotal role played by the protruding stalk composed of subunits Rpo4 and Rpo7 in the lattice contacts. Aided by MALLS (multi-angle laser light scattering), we have initiated the biophysical characterization of the recombinantly expressed and purified subunit Rpo13, a necessary step towards the understanding of Rpo13's role in archaeal transcription.

Protein–DNA interactions at theSulfolobusspindle-shaped virus-1 (SSV1) T5 and T6 gene promoters

Ultraviolet irradiation upregulates transcription from the Sulfolobus spindle-shaped virus 1 (SSV-1) T5, Tind, and T6 genes promoters and also triggers viral DNA replication, but nothing is known about the proteins involved in this process. A notable feature of T5 and T6 promoters is that they contain 4 copies of a highly conserved DNA sequence 5′-ATAGATAGAGT-3′; 2 copies of this repeat are found in tandem upstream of the A-box, whereas 2 additional tandem copies span the initiator region from which transcription originates. By employing electrophoretic mobility gel-shift assays (EMSAs) and chemical modification interference analyses, I have identified a protein STRIP (SSV-1 T5/T6 region-interacting protein) in Sulfolobus shibatae extract that binds specifically to this sequence. Unique to S. shibatae, STRIP induces a 28° bend in DNA. Surprisingly, despite the fact that STRIP binding masks the initiator region and can potentially interfere with preinitiation complex assembly, it does not appear to effect transcription driven from T5 and T6 promoters in vitro. Based on these results, I discuss the potential roles of STRIP in T5 and T6 transcription and in initiating SSV-1 DNA replication.

Role of the Sulfolobus shibatae viral T6 initiator in conferring promoter strength and in influencing transcription start site selection

Archaeal promoters contain a TATA-box, an adjacent upstream TFB-recognition element (BRE), and a downstream initiator (INR) region from which transcription originates. While the contribution of A-box and BRE to promoter strength is well established, the role of DNA sequences within the INR region and its vicinity on transcription efficiency and start site selection remains unclear. Here, I demonstrate using the strong Sulfolobus shibatae viral T6 promoter that either substitution of its natural sequence from –17 and beyond with plasmid DNA or introduction of point transversion mutations at +3, –2, –4, and –5 positions reduce promoter strength dramatically, whereas +1, –1, and –2 mutations influence the transcription start site. These data therefore reveal that the INR region plays a role as important as the BRE and the A-box in T6 gene transcription. Key words: Archaea, transcription, initiator (INR), Sulfolobus shibatae, core promoter.