The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases

Single in-frame amber (UAG) codons are found in the genes encoding MtmB, MtbB, or MttB, the methyltransferases initiating methane formation from monomethylamine, dimethylamine, or trimethylamine, respectively, in certain Archaea. The crystal structure of MtmB demonstrated that the amber codon codes for pyrrolysine, the 22nd genetically encoded amino acid found in nature. Previous attempts to visualize the amber-encoded residue by mass spectrometry identified only lysine, leaving information on the existence and structure of pyrrolysine resting entirely on crystallography of a single protein. Here we report successful mass spectral characterization of naturally occurring pyrrolysine and the first demonstration of the amber-encoded residue in proteins other than MtmB. The sequencing of chymotryptic fragments from acetonitrile-denatured proteins by tandem mass spectrometry revealed the mass of the amber-encoded residue in MtmB, MtbB, and MttB as 237.2 +/- 0.2 Da. Fourier transform ion cyclotron resonance mass spectrometry produced an accurate measurement for the pyrrolysyl-residue as 237.1456 Da, within error limits of the predicted mass based on the empirical formula C(12)H(19)N(3)O(2). These measurements support the structure of pyrrolysine in MtmB as 4-methylpyrroline-5-carboxylate in amide linkage with the (epsilon)N of lysine but not the alternative structure in which the 4-substituent of the pyrroline ring is an amine group. The presence of pyrrolysine with statistically identical mass in all three methyltransferases is in keeping with the proposed direct incorporation of pyrrolysine into protein during translation of the UAG codon and suggests that MtbB and MttB may exploit the unusual electrophilicity of pyrrolysine during catalysis.     

Evidence for the existence in mRNAs of a hairpin element responsible for ribosome dependent pyrrolysine insertion into proteins

In the methanogenic archae Methanosarcina barkeri, insertion of pyrrolysine, the 22nd amino acid, results from the decoding of an amber UAG codon in the mRNA of monomethylamine methyltransferases (MtmB). Sequence comparisons combined with structural enzymatic and chemical probing on M. barkeri MtmB1 mRNA demonstrate the presence of a hairpin motif located immediately after the redefined UAG codon. This structure of 86 nucleotides differs slightly from a proposal given in the literature and comprises four successive stems separated by three internal loops and closed by a large apical loop. Sequence alignments of MtmB mRNAs of different Methanosarcinacae reveal a conservation of the motif in both sequence and folding levels. The functional role of this motif as a signal leading to pyrrolysine insertion is discussed.     

Functional context, biosynthesis, and genetic encoding of pyrrolysine

In Methanosarcina spp., amber codons in methylamine methyltransferase genes are translated as the 22nd amino acid, pyrrolysine. The responsible pyl genes plus amber-codon containing methyltransferase genes have been identified in four archaeal and five bacterial genera, including one human pathogen. In Escherichia coli, the recombinant pylBCD gene products biosynthesize pyrrolysine from two molecules of lysine and the pylTS gene products direct pyrrolysine incorporation into protein. In the proposed biosynthetic pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by PylD. Structures of the catalytic domain of pyrrolysyl-tRNA synthetase (archaeal PylS or bacterial PylSc) revealed binding sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now being exploited as an orthogonal pair in recombinant systems for introduction of useful modified amino acids into proteins.     

Misacylation of pyrrolysine tRNA in vitro and in vivo

Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.     

Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases

Methanogenesis from trimethylamine, dimethylamine or monomethylamine is initiated by a series of corrinoid-dependent methyltransferases. The non-homologous genes encoding the full-length methyltransferases each possess an in-frame UAG (amber) codon that does not terminate translation. The amber codon is decoded by a dedicated tRNA, and corresponds to the novel amino acid pyrrolysine in one of the methyltransferases, indicating pyrrolysine to be the 22nd genetically encoded amino acid. Pyrrolysine has the structure of lysine with the (epsilon)N in amide linkage with a pyrroline ring. The reactivity of the electrophilic imine bond is the basis for the proposed function of pyrrolysine in activating and optimally orienting methylamine for methyl transfer to the cobalt ion of a cognate corrinoid protein. This reaction is essential for methane formation from methylamines, and may underlie the retention of pyrrolysine in the genetic code of methanogens.     

Retroviral gag gene amber codon suppression is caused by an intrinsic cis-acting component of the viral mRNA.

In some type C retroviruses, translation of the pol gene appears to require translational suppression of the proximal gag amber codon. To identify the region of the viral nucleic acid responsible for synthesis of the pol gene products, a 300-base-pair DNA fragment containing the stop codon from a type C murine virus (AK virus) was inserted into the Escherichia coli lacZ gene such that the translational reading frame was maintained. Introduction of the resulting fusion gene into cells resulted in the suppression of the viral stop codon. As measured by beta-galactosidase production, suppression occurred at a frequency of approximately 10%. Suppression could occur in at least several vertebrate cell types and was not augmented by virus replication or the expression of viral gene products. This indicates that gag amber codon suppression does not require augmented levels of suppressor tRNA species.     

Two modes of amber codon read-through in vitro

Read-through translation of bacteriophage R17 amB2 coat cistron carrying an amber mutation at the seventh codon was studied in vitro using the crude cell extract (S30) derived from an Escherichia coli nonsuppressor strain. Despite the presence of termination factors as well as ribosome-releasing factor (RRF) which prevent the read-through translation [M. Ryoji, J. W. Karpen, and A. Kaji (1981) J. Biol. Chem. 256, 5798-5801], synthesis of coat-like protein still persists at a low level in this system. Characterization of this protein by peptide fingerprinting and amino acid sequencing was performed to reexamine the generally accepted notion that it is produced by amino acid misinsertion to the amber mutation codon. The results indicated, however, that the major population of this coat-like protein is produced as a result of reinitiation of translation from the eighth codon. Read-through by amino acid misinsertion in this system becomes predominant only when the Mg2+ concentration is higher than 16 mM.     

Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.     

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λ c I and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.     

Constraints on codon context in Escherichia coli genes. Their possible role in modulating the efficiency of translation

The constraints on nucleotide sequences of highly and weakly expressed genes from Escherichia coli have been analysed and compared. Differences in synonymous codon spectra in highly and weakly expressed genes lead to different frequencies of nucleotides (in the first and third codon positions) and dinucleotides in the two groups of genes. It has been found that the choice of synonymous codons in highly expressed genes depends on the nucleotides adjacent to the codon. For example, lysine is preferably encoded by the AAA codon if guanosine is 3' to the lysine codon (AAA-G, P less than 10(-9)). And, on the contrary, AAG is used more often than AAA (P less than 0.001) if cytidine is 3' adjacent to lysine. Guanosine occurs more frequently than adenosine 5' to all the lysine codons (AAR, P less than 10(-5), i.e. NNG codons are preferred over the synonymous NNA codons 5' to the positions of lysine in the genes. The context effect was observed in nonsense and missense suppression experiments. Therefore, a hypothesis has been suggested that the efficiency of translation of some codons (for which the constraints on the adjacent nucleotides were found) can be modulated by the codon context. The rules for preferable synonymous codon choice in highly expressed genes depending on the nucleotides surrounding the codon are presented. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.