Figure 1.

Isolating RNA polymerase•ribosome complexes using different methods: (A) Sucrose gradient centrifugation. The top panel displays the sedimentation profiles of RNA polymerase alone (dashed blue line) and a stoichiometric mixture of RNA polymerase and ribosomes (solid red line) recorded at 280 nm. The two bottom panels display the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) result of each of the sucrose gradient fractions. The top panel shows the β/β′ region of the SDS-PAGE result of the gradient centrifugation of RNA polymerase (RNAP) alone, while the bottom presents the full gel of a mixture of RNA polymerase and ribosomes (RNAP + 70S, marker lane removed for clarity). (B) Glycerol gradient centrifugation. The panels are the same as in A. During ultracentrifugation, the complex of RNA polymerase and ribosome constantly re-equilibrates, causing the bound RNA polymerase to trail the ribosome in A and B. (C) Size exclusion chromatography. The top panel shows the elution profiles of a mixture of RNA polymerase and ribosomes (solid red line) and of RNA polymerase alone (dashed blue line; for comparison, the absorption is increased by 160-fold) from a 10/30 Superdex 200 column. (D and E) Capturing His-tagged RNA polymerase•ribosome complexes on a Ni-sepharose spin column. (D) SDS-PAGE results of all fractions, i.e. flowthrough (FT), washes with 0, 10, and 40 mM imidazole, and elution with 300 mM imidazole. (E) SDS-PAGE results of the first 300 mM imidazole elution step from Ni²⁺ affinity binding experiments. Various amounts of RNA polymerase and ribosomes are either loaded together or sequentially—first RNA polymerase (‘1st Load’), followed by a stoichiometric amount of ribosomes (‘2nd Load’). These experiments are performed in the presence of 30 mM KCl and 250 mM KCl. (F) RNA polymerase binding to ribosomes in the presence of 10 mM phosphate buffer (PO₄), 20 μM bovine serum albumin (BSA), a 14-nucleotide long RNA (RNA) at 5 μM, or 80 μg of poly(U)-RNA.