Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16S RNA

Abstract

Any base change at position 530 introduced into Escherichia coli on a multicopy plasmid leads to cell death [Powers & Noller (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1042-1046]. It was suggested that these mutants cannot carry out chain elongation. To define more precisely the function of base 530, we have studied ribosomes in which G530 was mutated to U530. In vivo, U530 16S rRNA was incorporated into 30S subunits and could combine with 50S to make 70S ribosomes. 16S rRNA in vitro transcripts containing U530 were assembled into 30S ribosomes, and their activity was tested in defined steps of protein synthesis. Mutant 30S ribosomes were as active as wild-type in poly(U)-dependent poly(Phe) synthesis, P- and A-site tRNA binding, and 30S initiation complex formation. 30S initiation complexes, in the presence of 50S, could react with puromycin like the wild-type. The rate, extent, and position of cross-linking of AcVal-tRNA in the P site to 16S RNA were identical in mutant and wild-type ribosomes. Although there appeared to be no defect in 70S initiation complex formation or in direct A-site binding of Phe-tRNA dependent on poly(U), U530 30S ribosomes were nevertheless defective in carrying out synthesis of fMet-Val dipeptide using natural mRNA. Mutant 30S ribosomes were also refractory to streptomycin-induced misreading although no misreading was observed in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

Testing constraints on rRNA bases that make nonsequence-specific contacts with the codon•anticodon complex in the ribosomal A site

“…These data confirm the work of others (Santer et al 1993) suggesting that G530 and A1492 mutations strongly reduce ribosome activity. As shown in Figure 1A, the structure of bacterial ribosome containing a cognate codon-anticodon complex shows that G530 and A1492 pair through an N1-HÁÁÁN1 hydrogen bond and that they interact with the middle codon-anticodon base pair with their 29-OH groups (Ogle et al 2001).…”

“…Any change to G530 or A1492 of the 16S rRNA confers a dominant lethal phenotype in E. coli (Powers and Noller 1990;Santer et al 1993;Yoshizawa et al 1999;Abdi and Fredrick 2005). These mutant rRNAs incorporate into 30S and 70S ribosomes (Powers and Noller 1990;Santer et al 1993;Abdi and Fredrick 2005) and assemble into polysomes (Powers and Noller 1990), but they translate natural mRNAs significantly less efficiently than do wild-type ribosomes (Santer et al 1993).…”

“…They realized that exchanging bases at 530 and 1492 (G530A and A1492G) would not disturb the geometry of this interaction. They predicted that a mutant ribosome with such an exchange mutation should be functional although each single mutant ribosome is inactive (Powers and Noller 1990;Santer et al 1993;Yoshizawa et al 1999;Abdi and Fredrick 2005). We decided to test this prediction using a dedicated ribosome system (Lee et al 1997).…”

16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: Deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases

“…No difference in exponential growth rates between wildtype and rsuA-deficient cells were found under laboratory conditions employing a range of temperatures in both rich and minimal media+ This was also true for RluA, RluC, and TruA in E. coli (Table 2), and is also the case in eukaryotes where deletion of specific guide RNAs blocks formation of the specified ⌿, but has no effect on cell growth or metabolism (Ofengand & Fournier, 1998)+ So far, only inactivation of RluD has a strong growth inhibitory effect (Table 2) Although the location of ⌿516 at the base of the highly conserved "530" loop , which is known to be involved in the fidelity of codon recognition (summarized in Santer et al+, 1993), is highly suggestive of a functional role in the small subunit of the ribosome, the location is not generally conserved in other organisms+ A homologous ⌿ exists in Bacillus subtilis (Wrzesinski et al+, 1995a;, and the existence of an ORF in Haemophilus influenzae with 73% homology to RsuA suggests the pres-FIGURE 5. Alignment of 111 Motif II sequences of the ⌿ synthase superfamily+ The 14 amino acids of Motif II are aligned and grouped according to families and subgroups within the families+ The subgroups are organized to highlight certain patterns of variations within the motif but do not indicate definitive functional or evolutionary groupings, although CLUST-ALX, NJplot, BLAST2 results, and functional information were considered when making the subgroups+ The motif consensus shown above the sequences summarizes the sequence patterns but does not necessarily indicate all pattern variations+ Motif conventions are as follows+ Single capital letters: invariant or nearly invariant; lower case letters: highly conserved; letter pairs indicate the two most common amino acids at that position; $: usually one of ILVM; ϩ: charged+ The existence of additional sequences in different organisms with exactly the same 14 amino acid sequence in Motif II are indicated by the numbers in parentheses+ The additional sequences are: TRUB ECOLI orthologs: TRUB_YEREN (sp:O34273); TRUB_ HAEIN (sp:P45142)+ TRUB_SCHPO ortholog: TRUB2/SCHPO (em:1319405)+ DKC1_HUMAN orthologs: CBF5_EMENI (sp:O43100); CBF5_YEAST (sp:P33322); CBF5_ASPFU (sp:O43102); CBF5_CANAL (sp:O43101); CBF5_KLULA (sp:O13473); DKC1_RAT (sp:P40615); NO60_DROME (sp:O44081)+ RLUD_ECOLI orthologs: RLUD_HAEIN (sp:P44445); RLUD_PSEAU (sp:P33640); RLUD/RICPR (em:1343115)+ The B. subtilus YTZF/BASCU protein sequence is a reconstruction of a probable frameshift mutation that fuses two adjacent protein sequences, YTZF_BACSU (O32068) and YTZG_ BACSU (O32069)+ There is also a human ortholog to the YD36_YEAST subgroup of the RluA family (tr:Q92939) that is not listed in the figure because it is a C-terminal partial sequence with the Motif II region still unsequenced+ The database record accession numbers are taken from a variety of databases abbreviated as sp: SWISS-PROT; pi: PIR; tr: TREMBL, em: EMBL; gb: Genbank; dd: DDBJ+ The citations to the original sequence papers can be found within the database records+ Locus names were taken from SWISS-PROT or are provisional designations devised as described in Materials and Methods+ When no ortholog was obvious and no other name was available, a generic family name was given to the protein as a temporary identifier, that is, RLUX or RSUX+ The SWISS-PROT organism codes used are ACICA: Acinetobacter calcoaceticus; AQUAE ence of an equivalent ⌿ in that organism+ On the other hand, Halobacter halobium does not have a homologous ⌿ and may not have any ⌿ at all in its SSU RNA …”

Free N‐Acetylneuraminic Acid in Tissues in Salla Disease and the Enzymes Involved in Its Metabolism

“…This enzyme was assayed using (2-3)-linked and (2-6)-linked N-a~etylneuraminyl[l-~H]lactitol a s described [4]. Methoxyphenyl N-acetylneuraminic acid neuraminidase activity was assayed using the method of Santer et al [16]. Liver homogenates were made in distilled water using a Potter-Elvejhem homogenizer, using approximately 20 mg liver in 250 pl water.…”