Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

Abstract

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix.

Fig. 1. The anti-association activity of IF3.

The extent of 70S initiation complex formation was monitored as a function of time by light scattering after rapid mixing in the stopped-flow instrument of a volume containing 30S subunits, mRNA, GTP and initiation factors as indicated with a volume containing 50S subunits. Time traces obtained with no initiation factors added to the 30S subunits (Panel A), with only IF3 added (Panel B), with IF1 and IF2 added (Panel C) and with IF1, IF2, IF3 and [³H]fMet-tRNAfMet added (Panel D).

Fig. 2. Rate of initiation in the absence of IF3 monitored by di-peptide formation.

The extent of dipeptide formation was monitored as a function of time after rapid mixing in a quench flow instrument of a volume containing 30S subunits, mRNA, GTP, IF1 and IF2 with an equal volume containing 50S subunits. Initiator tRNA was present either in the 30S or in the 50S mix. Time curves obtained with [³H]fMet-tRNAfMet added with 30S (Panel A), [³H]fMet-tRNAfMet added with 50S (panel B).

Fig. 3. The effects of G-nucleotides on the association of 30S pre-initiation complex with 50S subunits.

The extent of 70S initiation complex formation was monitored as a function of time by light scattering after rapid mixing of pre-initiation 30S complexes with 50S subunits in a stopped-flow instrument. Traces obtained with GTP (Panel A) and GDP (Panel B).

Fig. 4. The effects of buffer composition on the rate of 70S initiation complex formation.

The extent of 70S complex formation was monitored as a function of time by light scattering after rapid mixing of mixture A containing dissociated 70S ribosomes together with IF1, IF3 and mRNA with mixture B containing IF2:GTP together with fMet-tRNA. Complex formation in polymix buffer (PM) with 3 mM of free Mg₂₊ (Panel A), in PM buffer with 7 mM of free Mg₂₊ (Panel B) and in PM buffer with 3 mM free Mg₂₊ plus 10 mM of PEP (Panel C).

Fig. 5. The effects of the level of magnesium on the rate of naked 70S formation formation from its subunits.

The extent of 70S complex formation was monitored as a function of time by light scattering after rapid mixing of mixture A containing 30S ribosomes with mixture B containing 50S subunits. 70S formation in polymix buffer (PM) with 3 mM of free Mg²⁺ (Panel A), in PM buffer with 7 mM of free Mg²⁺ (Panel B).

Fig. 6. Purity of His-Tagged IF2 (IF2_HT) as seen on 10% SDS PAGE.

Lanes 1 and 5 contain protein molecular weight marker. Lanes 2, 3, and 4 contain increasing amounts of IF2_HT.