Assembly of Xenopus Transcription Factor III A-5S RNA Complex

The regulation of Xenopus 5S rRNA gene expression involves multiple protein factors, among which is transcription factor III A (TFIIIA). This factor can be isolated as a protein-RNA complex. The assembly behavior of this complex was studied by sedimentation velocity and gel electrophoresis at pH 7.5 and 23 °C. The reaction boundary was monitored by the absorbance at 260 nm; thus, the shape of the boundary reflects mainly RNA or RNA-containing complexes. Values for the weight-average sedimentation coefficient (s_2o,w) change with protein-RNA concentration. At low concentrations, values of s_2o,w increase with increasing concentration. The extrapolated value for s_2o,w ^at infinite dilution is 5.2 S, the same value for free Xenopus rRNA under the same experimentation conditions. Furthermore, the same value of s_2o,w at a specific RNA concentration can be obtained either by dilution of a concentrated sample or by concentrating a diluted one. These results indicate that complex formation can be described by a reversible process. When the data are analyzed by computer fitting, the simplest model that fits the sedimentation data is that TFIIIA and RNA form a 1:1 complex which self-aggregates to a dimer. The sedimentation coefficients (s_2o,w)of PR ^and (PP)2 ^are 7.5 and 10.6 S, respectively, where PR and (PR)₂ are the 1:1 TFIIIA-RNA complex and its dimer, respectively. The protein-RNA interaction was also investigated by gel electrophoresis. The resolved components were identified by differential staining for protein and RNA on a single gel. One band corresponding to free 5S rRNA was detected in addition to two bands which stained for both protein and nucleic acids. The mass ratio of these two protein-RNA complexes was determined to be 2.3, implying that one complex is probably a dimer of the other. These electrophoretic results indicate that at low concentration TFIIIA and RNA exist as individual free entities. At higher concentration, they form a complex which dimerizes. These results are totally consistent with that of sedimentation. In summary, the Xenopus TFIIIA-RNA complex undergoes a reversible equilibrium of macromolecular assembly which is characterized as TFIIIA + RNA ⇌ TFIIIA-RNA complex ⇌ (TF-IIIA-RNA complex)₂.