Leader peptides on precursors of proteins inserted into membranes, or translocated through them, consist of amino acids sequences that generally play a role in protein targeting. However, the nucleotides encoding them are not typically involved in the regulation of precursor synthesis. In this study, we explored genetically the function of the 15-amino-acid pre-Cox2p leader peptide and the mRNA encoding it, specified by S. cerevisiae
mtDNA. We examined the effects of mutations on the synthesis and function of pre-Cox2p and on the synthesis of a reporter protein, Arg8p, whose coding sequence was translationally fused to COX2
codon 91. Our data clearly demonstrate that the translation of the COX2
mRNA depends upon the mRNA sequence of codons specifying the pre-Cox2p leader peptide, in addition to its previously known dependence upon mRNA-specific translational activation by Pet111p through the 5′-UTL (17
First, we found that deletion of 13 leader peptide codons (2 to 14) from the cox2::ARG8m reporter mRNA prevented synthesis of the reporter protein despite normal mRNA levels. However, ARG8m was expressed from the COX2 locus in the complete absence of any COX2 codons. These findings strongly suggest that the first 91 codons of the pre-Cox2p coding sequence contain antagonistic elements that control translation positively and negatively: the positively acting element includes sequences in the first 14 codons specifying the leader peptide, while the negatively acting element appears to be within codons 15 to 91.
Our further analysis of mutations within the leader peptide coding sequence indicates that the positively acting element is complex or multipartite. None of the smaller deletion mutations we made in this region produced phenotypes as strongly negative as the deletion of codons 2 to 14 (cox2-20). Deletion of codons 2 to 6 (cox2-27) caused both leaky respiratory and leaky Arg− phenotypes without affecting mRNA levels, demonstrating the importance of this region for translation. However, residual growth of the cox2-27 and cox2-27::ARG8m strains indicates that the leader peptide codons remaining in this mutation, codons 7 to 14, also have a positive effect on translation in the absence of codons 2 to 6.
The positively acting element embedded in the leader peptide coding sequence functions at the level of nucleotide sequence, not amino acid sequence, since a shift of the reading frame through this region left the mRNA sequence relatively untouched while changing the amino acid sequence and did not detectably affect translation. On the other hand, when we altered the mRNA sequence of codons 2 to 6 (in the absence of codons 7 to 13) without changing the amino acid sequence they encoded (cox2-41
), we completely eliminated translation. In this connection, it is noteworthy that the first six codons contain an 11-base sequence (AGAUUUAUUAA) that is also present one base upstream of the initiation codon in the COX2
5′-UTL. It is tempting to speculate that the striking direct repeat of these bases plays a role in the positive regulatory function of the first six codons. The repeats could also play a role in the stringent selection of the COX2
initiation site, which they bracket. The ARG8m
coding region lacks this sequence, yet is efficiently expressed in the absence of COX2
codons, indicating that repetition of this sequence in the coding region is not essential for general translation initiation or elongation. However, the sequence repeat could be important for antagonizing the negative element in downstream COX2
codons. A mutation altering the upstream copy of the sequence repeat in the 5′-UTL had a modest negative effect on COX2
) and greatly reduced cox2
expression (N. Bonnefoy, unpublished data), consistent with this possibility.
Two different but overlapping mutations in the leader peptide coding region behave similarly, suggesting that they affect the same mechanism. Translation of the cox2::ARG8m reporter was reduced to a similar degree both by deletion of codons 2 to 6 (cox2-27) and by a compound allele in which codon 6 was changed from AGA to CGU (both encoding R), and codons 7 to 10 (cox2-22) were deleted. Both of these alleles were suppressed by nucleotide substitutions clustered in the downstream part of the leader peptide coding region and, in the case of cox2-22, the first three codons specifying mature Cox2p. Interestingly, we selected two different missense substitutions affecting codon 11 as suppressors of both cox2-27 and cox2-22, supporting the idea that the two original mutations impaired translation by a similar mechanism. In addition, some intragenic suppressors of each of the mutations were silent third-position substitutions in the COX2 coding sequence, consistent with our other data showing the importance of nucleotide sequence.
Similar stem-loop structures are predicted for the COX2 mRNAs of the wild type, cox2-27, and cox2-22 in the sequence corresponding to wild-type codons 10 through 17. This stem appears to exist in vivo and to play a role in reducing the translation of the mutant mRNAs, since all but one of the intragenic suppressors of these mutations that increased translation also weakened the stem. Furthermore, translation was reduced by compensating site-directed mutations that restored pairing with cox2-27 suppressor substitutions. Indeed, generation of a more stable stem, by conversion of a U:A base pair to G:C, reduced expression to a level below that of cox2-27. These results demonstrate that unfolding of the stem is necessary to improve translation of downstream coding sequences in the absence of codons 2 to 6. One possible interpretation of these data is that the residual positively acting sequences in codons 7 to 14 must be unfolded to function in the absence of codons 2 to 6. An alternative interpretation is that in the absence of codons 2 to 6, a stable stem could strengthen the action of the negative element downstream of codon 14 (this stem-loop cannot solely comprise the negative element since cox2-27 mRNAs are translated better than cox2-20 and cox2-41 mRNAs which lack it). These are not mutually exclusive possibilities, considering the potential dynamics of mRNA structure during translation.
We identified two nuclear genes, PET111
, as dosage-dependent suppressors of cox2-27
. Pet111p is the COX2
mRNA-specific translational activator (32
), a rate-limiting factor in COX2
). Elevated Pet111p activity can also suppress mutations in both the mRNA 5′-UTL (31
) and the initiation codon (6
). Since a fraction of ribosomes are able to pass the leaky translational blocks caused by the cox2-27
mutations, increased initiation would be expected to improve gene expression.
MrpL36p was previously found to be associated with mitochondrial ribosomal large subunits (28
), suggesting that it may function during translation elongation. This protein, which we found to be essential for global mitochondrial gene expression, contains a central 80-residue region exhibiting recognizable similarity to the entire length of the bacterial L31 family of ribosomal proteins in a PSI-BLAST comparison. Little is known about L31, except that it is loosely associated with the large subunit of Escheridria coli
). Loose ribosomal association of MrpL36p could account for the unexpected isolation of a dosage-dependent suppressor encoding a component of the ribosome. Flanking the central L31-like region is a roughly 60-residue N-terminal sequence exhibiting no similarities to known proteins and a 60-residue C-terminal region with weak but nevertheless intriguing similarity in a PSI-BLAST analysis to a short region of E. coli
Ffh, a protein subunit of the signal recognition particle. This region of similarity lies within the Ffh M domain, which binds to both the 4.5S RNA and the signal sequence of membrane proteins (16
). Thus, one could speculate that MrpL36p might mediate regulatory interactions among the elongating ribosome, positive and negative elements in the COX2
mRNA coding sequence, and the nascent pre-Cox2p polypeptide to coordinate synthesis and translocation of the pre-Cox2p N-tail domain through the inner membrane.
The role of the pre-Cox2p leader peptide amino acid sequence in controlling synthesis and membrane insertion of the protein remains enigmatic. The leader peptide sequences from several fungal and plant species are not strongly conserved (20
), and animal forms of Cox2p lack it entirely. While a previous study indicated that the leader peptide causes membrane association of a passenger protein (21
), our present findings demonstrate that several amino acid sequences, which were generated by frameshifts and various peptide lengths, can effectively carry out this and/or any other steps necessary for cytochrome oxidase assembly. For example, while the wild-type leader peptide is 15 residues long with an acidic group at the third position, we observed normal Cox2p accumulation and respiratory growth in a strain whose frameshifted leader peptide is 5 residues long, lacks an acidic group, and has a basic group at the second position (cox2-43R1
Nevertheless, our genetic analysis indicates that the leader peptide amino acid sequence is not completely unconstrained with respect to function in cytochrome oxidase assembly. One allele, cox2-43, allowed synthesis of the reporter protein and Cox2p at reduced rates, but prevented Cox2p from assembling into active cytochrome oxidase. The leader peptide encoded by cox2-43 is strikingly different from the wild type and the other functional sequences we generated, in that it has positively and negatively charged residues (KD) just upstream of the processing site. Spontaneous pseudorevertants of cox2-43 all had identical deletions of the six bases encoding these charged residues, which allowed efficient cytochrome oxidase assembly and greatly improved translation of the reporter protein. It is striking that the pseudorevertant encoding an active form of pre-Cox2p also increased mRNA translation.
We propose that the positive and negative translational regulatory elements specified within the first 91 codons of COX2 could function to ensure that continued translation of the mRNA occurs only if the nascent N terminus has successfully begun the process of membrane insertion leading to cytochrome oxidase assembly. While we cannot yet present a detailed model for how this feedback control mechanism might work, MrpL36p and Pet111p could function to convey information about the state of the nascent pre-Cox2p N terminus to the translating ribosome at the point where it encounters the negative element. We have also isolated dominant nuclear suppressors of both leader peptide mutations cox2-22 and cox2-27, which may identify other components of this system and lead to a better understanding of its mechanism. Passage through this element is likely to involve a dynamic interplay between alternative mRNA secondary structures and bound proteins.
Similar assembly feedback regulation of ATP synthase biogenesis could operate via pre-Atp6p, the only other yeast mitochondrial gene product with a leader peptide (30
). Such systems would resemble other translational feedback regulatory loops that couple the synthesis of specific components to the assembly of complexes. For example, translation of the chloroplast mRNA encoding cytochrome f
is coupled to assembly of the cytochrome b6/f
complex in Chlamydomonas
). In Caulobacter
, translation of the flagellin fljK
mRNA is regulated by assembly of the basal body-hook structure (1
). In this case, in which the flagellin is transported out of the cell by a type III secretion system, the regulation of fljK
translation depends on sequences in both the fljK
mRNA 5′-UTL and the first 9 codons of the structural gene. Finally, in the type III secretion system of Yersinia
, signals necessary for both the translation and the secretion of Yop proteins have been mapped to the first 15 codons and shown to function at the level of nucleotide sequence rather than amino acid sequence (2
). Thus, the regulatory system revealed by this study is likely to have its origins in the bacterial ancestors of mitochondria.