A temperature-sensitive mutant in the gene rplX for ribosomal protein L24 and its suppression by spontaneous mutations in a 23S rRNA gene of Escherichia coli.

A temperature-sensitive mutant with an altered ribosomal protein L24 was analysed. Revertant analysis showed that the temperature-sensitive growth was correlated with the altered protein. A DNA segment containing the mutant rplX gene was cloned and sequenced. The GGC codon for glycine at the amino acid position 84 of the protein was found to be altered to a GAC codon for aspartic acid. By transforming the rplX mutant with a plasmid carrying the rrnB operon and by selecting for temperature-resistant transformants we obtained two spontaneous suppressor mutants in the gene for 23S rRNA. DNA sequence analysis of the region corresponding to the 5' end of the 23S rRNA showed a C to T alteration at position 33 in both mutants and an additional A to G alteration at position 466 in one of them. The results suggest intimate interaction of protein L24 and the 5' end of 23S rRNA in vivo and support a secondary structure model of the 23S rRNA which brings these mutational points into a close contact.     

Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.     

一个母系遗传非综合征耳聋大家系mtDNA序列分析

通过分析本家mtDNA序列,探讨淮阴一非综合耳聋大家患病的分子遗传学机制。采用聚合酶链反应（PCR）扩增mtDNA与非综合征耳聋相关位点nt1555,nt7445的区域和人类种群研究的D－loop区,PCR－异源双链分析,PCR－RFLP、PCR产物克隆序列测定等技术对该家系进行了系统的研究。发现该家系中全部母系亲属有mtDNAA1555G突变,而家系中非母 个体,对照组（100例正常个体）的mtDNA1555位点均为A。该家系mtDNA7445位点无突变;该系属于Ⅱ型线性体;发现家系D－loop区存在未见报道的碱基插入。提示mtDNAA1555G位点突变可能是导致该家系患致聋的主要因素之一。遗传背景可能对家系疾病的表现存在一定程度的影响。     

Structural changes in the 530 loop of Escherichia coli 16S rRNA in mutants with impaired translational fidelity.

The higher order structure of the functionally important 530 loop in Escherichia coli 16S rRNA was studied in mutants with single base changes at position 517, which significantly impair translational fidelity. The 530 loop has been proposed to interact with the EF-Tu-GTP-aatRNA ternary complex during decoding. The reactivity at G530, U531 and A532 to the chemical probes kethoxal, CMCT and DMS respectively was increased in the mutant 16S rRNA compared with the wild-type, suggesting a more open 530 loop structure in the mutant ribosomes. This was supported by oligonucleotide binding experiments in which probes complementary to positions 520-526 and 527-533, but not control probes, showed increased binding to the 517C mutant 70S ribosomes compared with the non-mutant control. Furthermore, enzymatic digestion of 70S ribosomes with RNase T1, specific for single-stranded RNA, substantially cleaved both wild-type and mutant rRNAs between G524 and C525, two of the nucleotides involved in the 530 loop pseudoknot. This site was also cleaved in the 517C mutant, but not wild-type rRNA, by RNase V1. Such a result is still consistent with a more open 530 loop structure in the mutant ribosomes, since RNase V1 can cut at appropriately stacked single-stranded regions of RNA. Together these data indicate that the 517C mutant rRNA has a rather extensively unfolded 530 loop structure. Less extensive structural changes were found in mutants 517A and 517U, which caused less misreading. A correlation between the structural changes in the 530 loop and impaired translational accuracy is proposed.     

Interaction of translation initiation factor IF1 with the E. coli ribosomal A site

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.     

Arrangement of the sense and terminating codons of the template in the A-segment of human ribosomes from photocrosslinking data withe oligonucleotide derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3'-flanking sense codon or stop codon carrying a perfluoroarylazido group at G or U were used to study the position of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, UCC-recognizing tRNA(Phe) was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 or nucleotide A1825 in helix 44 close to the 3' end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem-loop segment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in various organisms.     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.     

A mutation in the GTPase domain of the large subunit rRNA is involved in the suppression of a -1T frameshift mutation affecting a mitochondrial gene in Chlamydomonas reinhardtii

The dum19 mutation isolated in Chlamydomonas reinhardtii is due to the deletion of one T at codon 152 of the mitochondrial cox1 gene sequence. Phenotypically, the dum19 mutant is characterized by a lack of cytochrome c oxidase activity and is unable to grow under heterotrophic conditions. A spontaneous pseudo-revertant that grows slowly in the dark was isolated from the dum19 mutant strain. A genetic and molecular analysis allowed us to demonstrate that the revertant phenotype is the consequence of two additional mutations that together act as a frameshift suppressor: an m mutation affecting a mitochondrial gene other than cox1 and an n mutation affecting a nuclear gene. On its own the n mutation does not act as a suppressor, whereas the m mutation very slightly compensates for the effect of the -1T mutation. Sequencing analysis showed that the m mutation affects the GTPase-associated domain of the large subunit (LSU) ofmitochondrial rRNA. Surprisingly, two substitutions, A1090 to G and A1098 to C, were found in the LSU rRNA of the revertant, the latter one being already present in the dum19 mutant strain itself. The A1090 to G substitution is thus involved in the suppression of the frameshift mutation, but it is not clear whether the change at position 1098 is also required for the expression of the suppressed phenotype. To our knowledge, this is the first example of a mutation in the GTPase-associated domain acting as a suppressor of a frameshift mutation.     

Mechanism of translation based on intersubunit complementarities of ribosomal RNAs and tRNAs

A universal rule is found about nucleotide sequence complementarities between the regions 2653-2666 in the GTPase-binding site of 23S rRNA and 1064-1077 of 16S rRNA as well as between the region 1103-1107 of 16S rRNA and GUUCG (or GUUCA) of tRNAs. This rule holds for all species in the living kingdoms except for two protista mitochondrial rRNAs of Trypanosoma brucei and Plasmodium falciparum. We found that quite similar relationships for the two species hold under the assumption presented in the present paper. The complementarity between T-loop of tRNA and the region 1103-1107 of 16S rRNA suggests that the first interaction of a ribosome with aminoacyl-tRNAEF-TuGTP ternary complex or EF-GGDP complex could occur at the region 1103-1107 of 16S rRNA with the T-loop-D-loop contact region of the ternary complex or the domain IV-V bridge region of the EF-GGDP complex. The second interaction should occur between the A-site codon and the anticodon loop or between the anticodon stem/loop of A-site tRNA and the tip of domain IV of EF-G. The above stepwise interactions would facilitate the collision of the region 1064-1077 of 16S rRNA with the region around A2660 at the alpha-sarcin/ricin loop of 23S rRNA. In this way, the universal rule is capable of explaining how spectinomycin-binding region of 16S rRNA takes part in translocation, how GTPases such as EF-Tu and EF-G can be introduced into their binding site on the large subunit ribosome in proper orientation efficiently and also how driving forces for tRNA movement are produced in translocation and codon recognition. The analysis of T-loops of all tRNAs also presents an evolutionary trend from a random and seemingly primitive sequence, as defined to be Y type, to the most developed structure, such as either 5G7 or 5A7 types in the present definition.     

A ribosomal ambiguity mutation in the 530 loop of E. coli 16S rRNA.

A series of base substitution and deletion mutations were constructed in the highly conserved 530 stem and loop region of E. coli 16S rRNA involved in binding of tRNA to the ribosomal A site. Base substitution and deletion of G517 produced significant effects on cell growth rate and translational fidelity, permitting readthrough of UGA, UAG and UAA stop codons as well as stimulating +1 and -1 frameshifting in vivo. By contrast, mutations at position 534 had little or no effect on growth rate or translational fidelity. The results demonstrate the importance of G517 in maintaining translational fidelity but do not support a base pairing interaction between G517 and U534.     

Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction

Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA. As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance. A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool. A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA. In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations. These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome. This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop.     

A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.     

Mutations in MTO2 related to tRNA modification impair mitochondrial gene expression and protein synthesis in the presence of a paromomycin resistance mutation in mitochondrial 15 S rRNA

Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA.     

Mutational study of the rRNA in yeast mitochondria: functional importance of T1696 in the large rRNA gene.

Four intragenic suppressors of a mitochondrial mutation in the 21S rRNA gene have been characterized in S. cerevisiae. The determination of the nature of the nucleotide changes in the suppressor strains showed that a T at position 1696 in the large rRNA gene is essential for correct function of the mitoribosome. The importance of this specific nucleotide and the fact that this mitochondrial mutation can also be suppressed by a mutation in a nuclear gene are in good agreement with a rRNA-r protein interaction in this part of domain IV, which functional importance is demonstrated in vivo by our results.     

Corrections to the human mitochondrial ribosomal RNA sequences

Two new errors and one consensus change were identified in the human mitochondrial Cambridge consensus sequence. The errors are an A to G substitution at nucleotide 750 in the 12S rRNA gene and a single nucleotide deletion at nt 3107 in the 16S rRNA gene. The consensus change is nt 2706 AG in the 16S rRNA gene.     

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells

To know the nature and mechanisms of spontaneous mutations in mitochondrial DNA (mtDNA), we determined, by direct cycle sequencing, the nucleotide sequence of the 3' terminal region of the mitochondrial 16S rRNA gene from chloramphenicol-resistant (CAP-R) mutants isolated in Chinese hamster V79 cells. Four different base substitutions were identified in common for the six CAP-R mutants. All mutations were heteroplasmic. One A to G transition was mapped at a site within the putative peptidyl transferase domain, the target region for chloramphenicol, and one G to A transition and two T to G transversions were located within the two different segments which form the stems of the hairpin loop structures attached to this key domain in the predicted secondary structure of 16S rRNA. The mutations detected in this study do not map to the same sites where CAP-R mutations were found previously in mammalian cells. Allele specific-PCR analyses revealed that all four mutations occurred on a single mutant-DNA molecule, but not on several ones independently. Together with the other previous reports, our data suggest that spontaneous mtDNA mutations may not be caused exclusively by oxidative DNA damage at least in 16S rRNA gene.     

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.     

A Three Nucleotide Signature Sequence in Small Subunit rRNA Divides Human Giardia in Two Different Genotypes

ABSTRACT. The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis , obtained from humans in The Netherlands (AMC-4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland-1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5'end of the 16S rRNA. The Portland-1 - Bris/83/HEPU/106 type has GCG in position 22–24, while AMC-4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are "signature" sequences, which divide Giardia isolates into two different groups.