Figure 3. An RNA with two zip code... | Rockefeller University Press

$Figure 3. An RNA with two zip code elements induces multimerization of SHE core complexes. (A–D) Dynamic light scattering (DLS) analysis and sucrose density gradient centrifugation of SHE core complexes with different RNA constructs. An RNA containing two zip code elements (B) induced the formation of much bigger complexes than RNA with only one zip code element (A and C). The increased hydrodynamic radius (Rh; numbers given in nanometer) and the faster sedimentation indicate multimerization of SHE core complexes in the presence of 2× ASH1-E3-77-tRNA. Furthermore, the broad distribution of complexes in the gradient suggests that a range of complex sizes was formed. No stable RNA complex formation was detected with HIV-1 TAR-tRNA (D), and only smaller subcomplexes co-migrated in the sucrose gradient. Fractions were analyzed by SDS-PAGE (proteins) with Coomassie blue staining and agarose gel electrophoresis (RNA) with GelRed staining. Schematics of the RNA constructs are shown on the right. Distinct features are color coded: ASH1-E3-77 (red), HIV-1 TAR (black), and three-way junction and tRNA (gray). Red dotted lines mark fractions 6 and 7, which contained the co-complex with a single zip code element (A).$

Figure 3.

An RNA with two zip code elements induces multimerization of SHE core complexes. (A–D) Dynamic light scattering (DLS) analysis and sucrose density gradient centrifugation of SHE core complexes with different RNA constructs. An RNA containing two zip code elements (B) induced the formation of much bigger complexes than RNA with only one zip code element (A and C). The increased hydrodynamic radius (Rh; numbers given in nanometer) and the faster sedimentation indicate multimerization of SHE core complexes in the presence of 2× ASH1-E3-77-tRNA. Furthermore, the broad distribution of complexes in the gradient suggests that a range of complex sizes was formed. No stable RNA complex formation was detected with HIV-1 TAR-tRNA (D), and only smaller subcomplexes co-migrated in the sucrose gradient. Fractions were analyzed by SDS-PAGE (proteins) with Coomassie blue staining and agarose gel electrophoresis (RNA) with GelRed staining. Schematics of the RNA constructs are shown on the right. Distinct features are color coded: ASH1-E3-77 (red), HIV-1 TAR (black), and three-way junction and tRNA (gray). Red dotted lines mark fractions 6 and 7, which contained the co-complex with a single zip code element (A).

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