Results: 5

1.
Figure 1

Figure 1. From: Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii.

Secondary structure of H.volcanii SRP RNA. Helices are numbered from 1 to 8 starting from the 5′-end. Numbering of residues is in increments of 10, as indicated by dots and numbers. Watson–Crick paired residues are connected by lines and G–U interactions by circles. A phylogenetically supported tertiary interaction (ter) between the loops of helices 3 and 4 is indicated.

Irit Tozik, et al. Nucleic Acids Res. 2002 October 1;30(19):4166-4175.
2.
Figure 4

Figure 4. From: Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii.

Reconstitution of H.volcanii SRP. Haloferax volcanii SRP19 and SRP54 were combined with SRP RNA either separately or together and the resulting complexes were examined by sucrose density gradient centrifugation. The levels of each component in the fractions collected from each gradient were determined densitometrically following electrophoresis and are presented as units. (A) Gradients prepared in 1 M KCl. (B) Gradients prepared in low salt. In both (A) and (B), the upper panels present the level of SRP19 in fractions collected from gradients containing SRP19 alone (filled circles), as well as the levels of SRP RNA (open circles) and SRP19 (filled squares) in fractions collected from gradients to which both components were added together. The middle panels in (A) and (B) present the levels of SRP54 in fractions collected from gradients containing SRP54 alone (filled circles), as well as the levels of SRP RNA (open circles) and SRP54 (filled squares) in fractions collected from gradients to which both components were added together. The lower panels in (A) and (B) present the levels of SRP RNA (filled circles), SRP19 (filled squares) and SRP54 (open circles) in fractions collected from high and low salt containing gradients containing all the components, respectively. In the lower panel of (B), the level of SRP RNA (open squares) in fractions collected from a low salt gradient to which SRP RNA alone was added is also shown.

Irit Tozik, et al. Nucleic Acids Res. 2002 October 1;30(19):4166-4175.
3.
Figure 5

Figure 5. From: Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii.

Immunodetection of H.volcanii SRP54. (A) Aliquots of wild-type E.coli cells, IPTG-induced E.coli cells transformed with plasmid pET-Hv54 encoding for H.volcanii SRP54, or Ni–NTA-purified H.volcanii SRP54 were probed with polyclonal antiserum raised against the H.volcanii SRP54. The specificity of antibody labeling of the bacterially expressed archaeal protein was confirmed by the failure of normal rabbit serum (NRS) to label such a band in E.coli cell extracts. Similarly, the polyclonal antibodies recognized SRP54 in an extract of H.volcanii. Again, NRS failed to label any such band. (B) Aliquots of H.volcanii cells, isolated membranes, the S100 cytoplasmic fraction and isolated ribosomes were probed with anti-H.volcanii SRP54 antibodies. The S100 and ribosomal fractions were prepared as described by Ban et al. (39). In both (A) and (B), the arrow depicts the position of the labeled 54 kDa protein recognized by the antibodies, while molecular weight markers shown on the right denote the 250, 150, 100, 75, 50, 37 and 25 kDa positions.

Irit Tozik, et al. Nucleic Acids Res. 2002 October 1;30(19):4166-4175.
4.
Figure 2

Figure 2. From: Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii.

Purification of H.volcanii SRP19. (A) Alignment of H.volcanii (H. vol) SRP19 with SRP homologs from Halobacterium sp. NRC-1 (H. NRC), Archeoglobus fulgidus (A. ful), Methanococcus jannaschii (M. jan), Pyrococcus abyssi (P. aby), Sulfolobus solfataricus (S. sol), Thermoplasma volcanium (T. vol) and Homo sapiens (H. sap). Lines are placed above every tenth residue in the H.volcanii sequence, while H.volcanii SRP19 residues discussed in the text are denoted by an arrow and the residue number. Homology of the sequences is shown below each residue, with the colon depicting identity and the period depicting similarity. (B) The relative proportions of basic and acidic amino acid residues in archaeal SRP19 proteins are shown. The same species as addressed in (A) are shown. Arginine residues are depicted in black, lysine residues in grey, aspartic acid residues in a speckled pattern and glutamic acid residues in a scratch mark pattern. (C) Escherichia coli BL21(DE3) cells were transformed with pET-Hv19, encoding for H.volcanii SRP19, and induced with 1 mM IPTG for a period of 3 h. Lane 1, wild-type cells; lane 2, uninduced transformed cells; lane 3, induced transformed cells. Molecular weight markers are shown on the right and correspond to 116, 66, 45, 35, 25, 18.4 and 14.4 kDa, while the arrow depicts the position of SRP19. The supernatant of induced cells was applied to Q-Sepharose and eluted with a gradient of NaCl. Lane 4, high spin supernatant; lane 5, Q-Sepharose eluted protein. Molecular weight markers shown on the right correspond to 43, 29, 18.4, 14.4, 6.2 and 3 kDa.

Irit Tozik, et al. Nucleic Acids Res. 2002 October 1;30(19):4166-4175.
5.
Figure 3

Figure 3. From: Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii.

Purification of H.volcanii SRP54. (A) Alignment of the M domains of H.volcanii (H. vol) SRP54 and E.coli (E. col) Ffh. Haloferax volcanii SRP54 residues discussed in the text are denoted by an arrow and the residue number, while lines are placed above each tenth residue of the H.volcanii sequence. Asterisks denote those E.coli Ffh residues in contact with SRP RNA. Homology of the sequences is shown below each residue, with the colon depicting identity and the period depicting similarity. (B) The relative proportions of basic and acidic amino acid residues in archaeal SRP54 proteins are shown. A.ful, Archaeoglobus fulgidus; H.NRC, Halobacterium sp. NRC-1; H.vol, Haloferax volcanii; M.jan, Methanococcus jannaschii; M.the, Methanococcus thermoautotrophicum; P.hor, Pyrococcus horikoshii; S.aci, Sulfolobus acidocaldarius; T.aci, Thermoplasma acidophilum. Arginine residues are depicted in black, lysine residues in grey, aspartic acid residues in a speckled pattern and glutamic acid residues in a scratch mark pattern. (C) Escherichia coli BL21(DE3) cells were transformed with pET-Hv54, encoding for His6-tagged H.volcanii SRP54 and induced with 0.5 mM IPTG for a period of 3 h. The supernatant of induced cells was incubated with Ni–NTA resin and eluted with buffer containing 0.5 M imidazole. Lane 1, wild-type cells; lane 2, uninduced transformed cells; lane 3, induced transformed cells; lane 4, high spin supernatant; lane 5, Ni–NTA-bound proteins. Molecular weight markers are shown on the right and correspond to 250, 150, 100, 75, 50, 37 and 25 kDa, while the arrow depicts the position of SRP54.

Irit Tozik, et al. Nucleic Acids Res. 2002 October 1;30(19):4166-4175.

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