A single mutation in the 15S rRNA gene confers non sense suppressor activity and interacts with mRF1 the release factor in yeast mitochondria

Abstract

We have determined the nucleotide sequence of the mim3-1 mitochondrial ribosomal suppressor, acting on ochre mitochondrial mutations and one frameshift mutation in Saccharomyces cerevisiae. The 15s rRNA suppressor gene contains a G633 to C transversion. Yeast mitochondrial G633 corresponds to G517 of the E.coli 15S rRNA, which is occupied by an invariant G in all known small rRNA sequences. Interestingly, this mutation has occurred at the same position as the known MSU1 mitochondrial suppressor which changes G633 to A. The suppressor mutation lies in a highly conserved region of the rRNA, known in E.coli as the 530-loop, interacting with the S4, S5 and S12 ribosomal proteins. We also show an interesting interaction between the mitochondrial mim3-1 and the nuclear nam3-1 suppressors, both of which have the same action spectrum on mitochondrial mutations: nam3-1 abolishes the suppressor effect when present with mim3-1 in the same haploid cell. We discuss these results in the light of the nature of Nam3, identified by 1 as the yeast mitochondrial translation release factor. A hypothetical mechanism of suppression by "ribosome shifting" is also discussed in view of the nature of mutations suppressed and not suppressed.

Keywords: 15S rRNA; frame shift; informational suppressors; nonsense; yeast.

Figure 1. FIGURE 1: Nucleotide sequence of suppressed and non-suppressed frameshift mutations in the RNA-maturase coding part of the second intron of the cytochrome b gene.

Note that both mutations create double ochre stop codons, one immediately after the addition of adenine (M3041) and the other after a shift of nine amino acids after the addition of adenine (G5006). Numbering like in table 1.

Figure 2. FIGURE 2: Physical mapping of the mim3-1 suppressor.

The restriction map and position of 15s rRNA and COX1 (subunit I of cytochrome oxidase) genes are shown at the top. tsl: locus involved in tRNA maturation. The KG01 rho^- petite, carrying the mim3-1 mutation, was subjected to ethidium bromide mutagenesis and sub-cloned twice in the absence of the drug. A few hundred colonies were tested for suppression of the M3041 target mutation. + and - indicates suppression or its absence. Most of the sub-clones did not suppress any longer; 24 rho^- sub-clones were further analysed by restriction (only a few sites are shown) and blotting with a probe constituted by the KG01 mitochondrial DNA. The lengths and positions of tandem repeats retained in mtDNA of three discriminating petites are represented by thick bars indicating that the suppressor is located in the 15s rRNA gene.

Figure 3. FIGURE 3:

(A) Nucleotide sequence of the mim3-1 region in the 15S rRNA. The nucleotide sequence of 629-754 region in the mitochondrial 15S rRNA of various strains is shown. Sequences are from: 1-; 2: ; 3: ; 4 and 5: . The position 633 is the only one mutated in the ochre suppressors MSU1 and mim3-1. The underlined bases are engaged in a stem structure. (B) Position of mim3-1 on the 530 stem-loop secondary structure. On the right the structure of yeast mitochondrial 15s rRNA is shown which is homologous to the 530 bacterial one, numbered from 5'. The G633 which is replaced by C in mim3-1 and by A in MSU1 suppressors is boxed. On the left the 530 stem-loop structure of the 15S rRNA in Eubacteria is shown with numbering according to E.coli. The substitution of G530 (circled) by either C, A or U is lethal in E.coli . Note that the boxed triplet in both structures is complementary and may interact in the tertiary structure according to .

Figure 4. FIGURE 4: Haploid cells carrying both mim3-1 and nam3-1 suppressors are respiratory deficient.

Fresh cultures of different strains are streaked on YPGA (glucose) and N3 (glycerol) plates and incubated three days at 28°C. All of these strains are isogenic: the nucleus is from JC8 and the mitochondria are from 777-3A. The nuclear genotype is given in brackets (with the nuclear one at the left and the mitochondrial at the right) with N+: NAM3⁺, n1: nam3-1; n2: nam3-2, n3: nam3-3; m: mim3-1; M: M3041.