DYNAMICS OF IN-VITRO ASSEMBLY OF 16-S RIBOSOMAL-RNA INTO 30-S RIBOSOMAL-SUBUNITS

One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly. As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly. One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0°C effects). At 30°C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2=<5 min at 30°C); (2) slow (t1/2=15 to 30 min at 30°C); (3) delayed slow (t1/2=30 to 60 min at 30°C). Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly. These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5′ to 3′ polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3′ major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.