Figure 5.

Both Brx1 and Ebp2 assemble early into pre-ribosomes. (A) KM1725 (myc-BRX1) cells were grown at 25°C to mid-log phase, then cell extracts were prepared and subjected to sucrose density gradient ultracentrifugation to resolve ribosomal subunits, monoribosomes and polyribosomes. A ribosomal profile was determined by measurement of OD₂₅₄ of gradient (7–47%) fractions. Each fraction was collected and subjected to SDS–PAGE and western analysis using antibodies against Ebp2 and the myc epitope. (B) Both Ebp2 and Brx1 associate with 90S pre-ribosomes containing 35S pre-rRNA, as well as 66S pre-ribosomes containing 27S pre-rRNAs. TAP-tagged Ebp2 and Brx1 were used to affinity-purify pre-ribosomes. Pre-RNAs contained in these pre-rRNPs were identified by primer extension and gel electrophoresis. The untagged parent strain S288c and Nob1-Tap were used as controls.