The function and structural characteristics of conserved regions within Escherichia Coli small subunit ribosomal RNA Public Deposited

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  • Ribosomes are multicomponent macromolecular particles and are essential for the survival of cells in all organisms. The ribosome's universal function is to catalyze polypeptide synthesis through translation of mRNA transcripts. Ribosomes from Escherichia coli, eubacterial organisms, have a sedimentation coefficient of 70S and are composed of 30S and 50S ribonucleoprotein subunits. The small ribosomal subunit is an assembly of 21 different proteins and a 16S ribosomal RNA. Within the 16S rRNA there are a few short stretches of universally conserved sequences spanning positions 517-533, 1394-1408, and 1492-1506. Clear functions for these sequence zones have not yet been assigned. Here I report a kinetic analysis of these highly conserved regions in the 16S rRNA and within the 30S ribosomal subunits. Binding affinity was measured in experiments that were based on protection from nuclease 51 digestion of short oligodeoxynucleotides hybridized to the designated regions. DNAs hybridized to regions 1400 and 1500 show significant differences in the apparent dissociation constants when measured in 30S particles as opposed to those found for 16S rRNA. Region 525 showed no difference in kinetic behavior. To further elucidate the functional and structural role played by the region centered about C1400 in 16S rRNA, a four nucleotide deletion was constructed within this region. The deletion was introduced by direct RNA manipulation using DNA/RNA hybridization, RNase H digestions, and ligation of the correct RNA fragments with T4 RNA ligase. I improved ligation efficiency of large RNA molecules by including a connector looped short DNA oligomer. Recycling products through phenyl boronate agarose (PBA-30) column also improved the efficiency of ligation. The mutagenized 16S rRNA fully reassembles into 30 particles and the altered 30S subunit possesses all of the normal ribosomal proteins. Altered ribosomes were functional in in vitro translation of MS2 mRNA. The altered ribosomes have lower translational activity relative to controls. Here I present indirect evidence suggesting that the decrease in the synthesis of MS2 coat proteins is the result of premature termination. The altered 16S RNA in ribosomes had an apparent dissociation constants with DNA probes comparable to those found for normal 16S rRNA. This suggest that the RNA is less flexible in the particle relative to normal 30S subunits. The deletion at 1400 did not have any effect on the physical properties of the 1500 region, as measured by DNA hybridization. A minor, but significant, effect on the 525 region was observed. A possible RNA/RNA interaction within the 30S particle is proposed to account for this observation.
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