Biomaterial Database

Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits. 1993

T Powers, and G Daubresse, and H F Noller

Sinsheimer Laboratories, University of California, Santa Cruz 95064.

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 degrees C effects). At 30 degrees 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 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees 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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D003080	Cold Temperature	An absence of warmth or heat or a temperature notably below an accustomed norm.	Cold,Cold Temperatures,Temperature, Cold,Temperatures, Cold
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006358	Hot Temperature	Presence of warmth or heat or a temperature notably higher than an accustomed norm.	Heat,Hot Temperatures,Temperature, Hot,Temperatures, Hot
D012269	Ribosomal Proteins	Proteins found in ribosomes. They are believed to have a catalytic function in reconstituting biologically active ribosomal subunits.	Proteins, Ribosomal,Ribosomal Protein,Protein, Ribosomal
D012270	Ribosomes	Multicomponent ribonucleoprotein structures found in the CYTOPLASM of all cells, and in MITOCHONDRIA, and PLASTIDS. They function in PROTEIN BIOSYNTHESIS via GENETIC TRANSLATION.	Ribosome
D012336	RNA, Ribosomal, 16S	Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis.	16S Ribosomal RNA,16S rRNA,RNA, 16S Ribosomal,Ribosomal RNA, 16S,rRNA, 16S
D015336	Molecular Probe Techniques	The use of devices which use detector molecules to detect, investigate, or analyze other molecules, macromolecules, molecular aggregates, or organisms.	Molecular Probe Technic,Molecular Probe Technics,Molecular Probe Technique,Technic, Molecular Probe,Technics, Molecular Probe,Technique, Molecular Probe,Techniques, Molecular Probe,Probe Technic, Molecular,Probe Technics, Molecular,Probe Technique, Molecular,Probe Techniques, Molecular