Abstract
We have investigated the highly conserved GAUCA sequence of small subunit ribosomal RNA. Within this region, the invariant nucleotides G1530 and A1531 of Escherichia coli 16 S rRNA were mutagenized to A1530/G1531. These base changes caused a lethal phenotype when expressed from a high copy number plasmid. In low copy number plasmids, the mutant ribosomes had limited effects when expressed in vivo but caused significant deficiencies in translation in vitro, affecting enzymatic tRNA binding, non-enzymatic tRNA binding, subunit association, and initiation factor 3 (IF3) binding. Mutant 30 S ribosomal subunits showed a 10-fold decrease in affinity for IF3 as compared to wild-type subunits but showed an increased affinity for IF3 when in 70 S ribosomes. Additionally, IF3 did not promote dissociation of 70 S ribosomes, which had mutated subunits as monitored by light-scattering experiments. However, extension inhibition experiments (toeprinting) showed that IF3 retained its ability to discriminate between initiator and elongator tRNAs on mutated subunits. The results indicate that the two functions of IF3, tRNA discrimination and subunit dissociation, are separable and that the invariant nucleotides are important for correct subunit function during initiation.
Highlights
Ribosomal RNA plays a significant role in the process of translation, and specific rRNA regions have been implicated in several translational functions [1, 2]
The GAUCA sequence immediately upstream of the anti-SD region is highly conserved in all three domains [8] and includes invariant nucleotides at positions 1530 and 1531 [9]
Expression of the 16 S rRNA Mutation G1530A/A1531G— Two invariant nucleotides (G1530/A1531) immediately 5Ј to the anti-Shine-Dalgarno sequence in 16 S rRNA were changed to A1530 and G1531 (Fig. 1) by oligonucleotide-directed mutagenesis
Summary
Ribosomal RNA (rRNA) plays a significant role in the process of translation, and specific rRNA regions have been implicated in several translational functions [1, 2]. The GAUCA sequence (nucleotides 1530– 1534 in Escherichia coli 16 S rRNA, see Fig. 1) immediately upstream of the anti-SD region is highly conserved in all three domains [8] and includes invariant nucleotides at positions 1530 and 1531 [9]. Present address: Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112. Based partially on the conserved nature of the region, Kossel et al [17] proposed that an interaction occurs between the GAUCA sequence and the 5Ј-end of 16 S rRNA as a discrete functional state during elongation, whereas Thanaraj and Pandit [18] have proposed that the GAUCA sequence functions as a translational enhancer by base pairing with a complementary sequence in mRNA upstream of the start codon. We found that expression of mutant 16 S rRNA affected several subunit functions in vitro, including initiation complex formation, subunit association, and IF3 binding
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