In vitro suppression of UGA codons in a mitochondrial mRNA

Although both prokaryotic and eukaryotic messenger RNAs can be easily translated in heterologous protein-synthesizing systems, attempts to achieve correct synthesis of mitochondrial proteins by translation of mitochondrial mRNAs in such systems have failed. In general, the products of synthesis are of low molecular weight and presumably represent fragments of mitochondrial proteins. These fragments display a strong tendency to aggregate. Explanations have included the use by mitochondria of codons requiring a specialized tRNA population and the fortuitous occurrence within genes of purine-rich sequences resembling bacterial ribosome binding sites. In addition, the long 5'-leader sequences present in many mitochondrial (mt) RNAs may also contribute to difficulties in mRNA recognition by heterologous ribosomes. Recent sequence analysis of human mtDNA suggests that the genetic code used by mammalian mitochondria deviates in a number of respects from the 'universal' code, the most striking of these being the use of the UGA termination codon to specify tryptophan. That this may also apply in yeast mitochondria has been shown by Fox and Macino et al., thus providing an obvious and easily testable explanation for the inability of heterologous systems to synthesize full-length mitochondrial proteins. We confirm this explanation and describe here the in vitro synthesis of a full-length subunit II of yeast cytochrome c oxidase in a wheat-germ extract supplemented with a partially purified mitochondrial mRNA for this protein and a UGA-suppressor tRNA from Schizosaccharomyces pombe.     

RNA editing by cytidine insertion in mitochondria of Physarum polycephalum

A corollary of the central dogma of molecular biology is that genetic information passes from DNA to RNA by the continuous synthesis of RNA on a DNA template. The demonstration of RNA editing (the specific insertion, deletion or substitution of residues in RNA to create an RNA with a sequence different from its own template) raised the possibility that in some cases not all of the genetic information for a trait residues in the DNA template. Two different types of RNA editing have been identified in mitochondria: insertional editing represented by the extensive insertion (and occasional deletion) of uridine residues in mitochondrial RNAs of the kinetoplastid protozoa and the substitutional editing represented by the cytidine to uridine substitutions in some plant mitochondria. These editing types have not been shown to be present in the same organism and may have very different mechanisms. RNA editing of both types has been observed in nonmitochondrial systems but is not as extensive and may involve still different mechanisms. Here we report the discovery of extensive insertional RNA editing in mitochondria from an organism other than a kinetoplastid protozoan. The mitochondrial RNA apparently encoding the alpha subunit of ATP synthetase in the acellular slime mould, Physarum polycephalum, is edited at 54 sites by cytidine insertion.     

The codon CUG is read as serine in an asporogenic yeast Candida cylindracea

Deviations from the universal genetic code have been reported for several microorganisms. Termination codons are used for coding some amino acids in Paramecium, Mycoplasma or Tetrahymena, and in Escherichia coli, the UGA termination codon is used to code for selenocysteine. In mitochondria, the changes of sense codons to termination codons or to codons encoding other amino acids have also been reported. Here we report another example of divergence from the universal code, this time in a non-spore-forming yeast Candida cylindracea, in which the universal codon for leucine, CUG, is used to code for serine. This conclusion is based on the observations that: (1) the amino-acid composition and the partial amino-acid sequences of an extracellular lipase from this yeast agreed with those deduced from the complementary DNA if CUG was assumed to specify serine; and (2) serine, but not leucine, was incorporated into a polypeptide in a cell-free translation system from this yeast in the presence of a synthetic CUG oligomer.     

Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.     

Converting nonsense codons into sense codons by targeted pseudouridylation

All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (Ψ), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to Ψ within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ΨAA and ΨAG code for serine and threonine, whereas ΨGA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code.     

Evolutionary nucleotide replacements in DNA.

With the increasing availability of analytical information on mRNA molecules, it is now possible to compare homologous nucleotide sequences from different organisms and to draw conclusions about their evolution. Such comparisons have shown that silent changes in codons occur more frequently than nucleotide replacements that produce changes in amino acid sequences (code-altering changes). Furthermore, there is an important difference between amino acid sequence comparisons and nucleotide sequence comparisons. The former show only differences in amino acid residues, but the latter show several types of differences when corresponding codons are compared. Single-base replacements may be degenerate (silent) or expressed as amino acid replacements. Two-base codon changes may be degenerate, single-base changes, or be visible as such. Three-base codon changes may be degenerate (involving serine), simulate either single-base or two-base changes or be visible as such. All nine types of change are found in comparisons of genes from the viruses phi X174 and G4. The relative numbers of these nine types as based on all possible interchanges between all 61 amino acid codons were listed by Holmquist et al. and are shown in Table 1. We discuss these results in the light of the significance of nucleotide changes in molecular evolution.     

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin （HA） cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.     

Independent transfer of mitochondrial chromosomes and plasmids during unstable vegetative fusion in Neurospora

The sporadic distribution of similar introns in organelle, nuclear ribosomal RNA and bacteriophage genes suggests that at least some of these introns are mobile genetic elements. Some plasmids in fungal mitochondria contain intron-like sequences and, like introns, they have scattered distributions within and among species. The occurrence and evolutionary importance of such horizontal transfer of DNA, not only between fungi, but among a wide range of organisms have been matters of much discussion. Here, we report experimental evidence for transfer of Neurospora mitochondrial plasmids from one mitochondrial genotype to another at high frequency during unstable vegetative cell fusion. Exchange of mitochondrial and nuclear genomes can also occur. These observations suggest that vegetative fusion may have a more important role in the mitochondrial genetic structure of natural populations than is generally thought, and may provide an explanation for the distribution of certain plasmids and possibly other mobile genetic elements.     

Does Paramecium primaurelia use a different genetic code in its macronucleus?

It has long been known that messenger RNAs (mRNAs) of ciliates and in particular of Paramecium are not translated well in heterologous in vitro translation systems. Recently, we have demonstrated for Paramecium primaurelia that this phenomenon results from the presence of well-defined blocking sites in the coding sequences of almost all mRNAs, and that these sites are an intrinsic feature of the primary as opposed to the secondary structure of the mRNAs. Here we show that both the gene and the mRNA for the G surface antigen of P. primaurelia contain numerous TAA and TAG codons scattered throughout their coding sequences. We propose that these codons do not represent termination codons in P. primaurelia but instead code for glutamic acid or glutamine and that the in vitro translation of Paramecium mRNAs is blocked by their presence.     

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

Molecular determinants and guided evolution of species-specific RNA editing

Most RNA editing systems are mechanistically diverse, informationally restorative, and scattershot in eukaryotic lineages. In contrast, genetic recoding by adenosine-to-inosine RNA editing seems common in animals; usually, altering highly conserved or invariant coding positions in proteins. Here I report striking variation between species in the recoding of synaptotagmin I (sytI). Fruitflies, mosquitoes and butterflies possess shared and species-specific sytI editing sites, all within a single exon. Honeybees, beetles and roaches do not edit sytI. The editing machinery is usually directed to modify particular adenosines by information stored in intron-mediated RNA structures. Combining comparative genomics of 34 species with mutational analysis reveals that complex, multi-domain, pre-mRNA structures solely determine species-appropriate RNA editing. One of these is a previously unreported long-range pseudoknot. I show that small changes to intronic sequences, far removed from an editing site, can transfer the species specificity of editing between RNA substrates. Taken together, these data support a phylogeny of sytI gene editing spanning more than 250 million years of hexapod evolution. The results also provide models for the genesis of RNA editing sites through the stepwise addition of structural domains, or by short walks through sequence space from ancestral structures.     

tRNA丰度是影响蛋白质二级结构形成的一个因素

从人和大肠杆菌蛋白质和DNA序列的统计分析，发现mRNA的对应一定蛋白质二级结构的m密码子片段(m=3，4，…，8)，当平均tRNA拷贝数高时(对于人高于11，对于大肠杆菌高于2．O)，偏好编码螺旋，而避免编码线团；当平均tRNA拷贝数低时，偏好性正好反过来．对于beta折叠，未发现和tRNA拷贝数有明显的统计关联．对大肠杆菌也研究了密码子频数与蛋白质二级结构的相关性，其间的关联比tRNA拷贝数与蛋白质二级结构的关联弱．因此，tRNA拷贝数与蛋白质二级结构的关联可能更为本质．机制目前尚不清楚．一种可能的解释是：tRNA丰度以某种方式影响了新生肽链的折叠，通过翻译精度的因素影响了蛋白质二级结构的形成．