Escherichia coli ribosomal subunits can be reconstituted in vitro under highly optimized conditions. These reconstitution systems have proven invaluable for the study of ribosomal subunit assembly. While E. coli ribosomal subunits can self-assemble in vitro there has been much speculation regarding the existence of extra-ribosomal assembly factors that act in functional subunit formation in vivo. Recently, a biochemical assay has been implemented to identify factors that facilitate a single, critical step in 30S subunit assembly in vitro. These studies have revealed that the DnaK (heat shock protein 70) chaperone system can facilitate 30S subunit assembly in vitro. The 30S subunits, formed in the presence of the chaperones under otherwise non-permissive conditions, are highly similar to 30S subunits formed under standard reconstitution conditions. It has become evident that the manner in which the "factor-assembled" 30S subunits are purified is critical for monitoring formation of functional ribosomal particles. Given that methodologies for in vitro reconstitution and functional analysis of ribosomal subunits have been described in detail previously, this manuscript will focus on isolation of functional 30S subunits that have been assembled in the presence of exogenous factors in vitro. Also, recent efforts toward understanding the roles of exogenous factors in 50S subunit and eukaryotic ribosome assembly will be briefly discussed.
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