Pyrrolysine is not hardwired for cotranslational insertion at UAG codons Alexandre Ambrogelly*, Sarath Gundllapalli*, Stephanie Herring*, Carla Polycarpo*†, Carina Frauer*‡, and Dieter So¨ ll*§¶ Departments of *Molecular Biophysics and Biochemistry and §Chemistry, Yale University, New Haven, CT 06520-8114 Contributed by Dieter So¨ll, December 28, 2006 (sent for review December 23, 2006) Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets barkeri PylRS and M. barkeri tRNAPyl and explore the fitness and acylated to its distinctive UAG suppressor tRNAPyl by the cognate coding response of this orthogonal tRNA for translation in E. coli. pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNAPyl in Results vivo by using the Pyl analog N-␧-cyclopentyloxycarbonyl-L-lysine. Nucleotides that Determine Fitness of tRNAPyl for Translation in E. coli. Forty-two Methanosarcina barkeri tRNAPyl variants were tested in To screen a large number of M. barkeri Fusaro tRNAPyl variants Escherichia coli for suppression of the lac amber A24 mutation; generated by mutagenesis, we made use of this tRNA’s ability to then relevant tRNAPyl mutants were selected to determine in vivo suppress the lac amber mutation A24 in a lacI–lacZ fusion binding to M. barkeri PylRS in a yeast three-hybrid system and to system (12, 13). Therefore, we transformed E. coli strain XAC/ measure in vitro tRNAPyl aminoacylation. tRNAPyl identity elements A24 with plasmid-borne copies of M. barkeri pylS and 42 mutant include the discriminator base, the first base pair of the acceptor pylT genes and grew the transformants in the presence of the Pyl stem, the T-stem base pair G51:C63, and the anticodon flanking analog N-␧-cyclopentyloxycarbonyl-L-lysine (Cyc), because Pyl is nucleotides U33 and A37. Transplantation of the tRNAPyl identity not commercially available (12). Suppression was quantitated by elements into the mitochondrial bovine tRNASer scaffold yielded measuring ␤-galactosidase activity (Table 1). The mutations chimeric tRNAs active both in vitro and in vivo. Because the covered all regions of the tRNAPyl; however, the anticodon was anticodon is not important for PylRS recognition, a tRNAPyl variant not altered because the suppression assay depended on the could be constructed that efficiently suppressed the lac opal U4 integrity of amber codon recognition. mutation in E. coli. These data suggest that tRNAPyl variants may decode numerous codons and that tRNAPyl:PylRS is a fine orthog- Discriminator Base and Acceptor Helix. Systematic mutation of the onal tRNA:synthetase pair that facilitated the late addition of Pyl nucleotides in the acceptor stem revealed that the discriminator to the genetic code. base G73 and the first base pair of the acceptor stem are major tRNAPyl identity elements. The G73A and G73U mutations orthogonal tRNA ͉ suppression ͉ tRNA identity ͉ pyrrolysyl-tRNA decreased suppression efficiency markedly. The base pair synthetase ͉ aminoacyl-tRNA synthetase G1:C72, conserved in all known tRNAPyl species, could be flipped with some loss of activity, yet replacement with a weaker ncorporation of noncanonical amino acids into proteins is an pair (G1:U72) or loss of the base pair (A1:C72) resulted in severe Iexciting and active research field. To date, Ͼ30 unnatural loss of suppression efficiency. These results suggest that the amino acids have been placed into proteins with high fidelity primary role of the first base pair is to ensure the proper mostly directed by the amber codon UAG (1, 2). The other two productive placement of recognition elements, such as the termination codons as well as enlarged codons (with 4–6 bases) discriminator base and the terminal adenosine. Mutation of the have also been used (e.g., refs. 3 and 4). The key step in this G2:C71 base pair to A2:U71 reduced suppression efficiency by process is the introduction of an orthogonal tRNA:aminoacyl- Ϸ50%. Conversion of the A3:U70 base pair into a G3:U70 base tRNA synthetase pair into the host protein synthesizing system. pair resulted in a drop of suppression efficiency. This was Such an orthogonal tRNA should not be recognized by any unexpected, as this G3 is found in wild-type M. barkeri MS endogenous aminoacyl-tRNA synthetase, whereas the orthogo- tRNAPyl. However, the MS tRNA also contains a C44U muta- nal synthetase should acylate solely the orthogonal tRNA with tion in the first nucleotide of the variable loop. Because both the unusual amino acid. independent mutations result in a decrease in suppression Less attention was devoted to incorporation of the nonca- efficiency (Table 1) and because the wild-type tRNAPyl species nonical amino acids selenocysteine (Sec) and pyrrolysine (Pyl) from both M. barkeri strains are equally good substrates for that arose from natural expansion of the genetic code (5). Sec, suppression, the two variations compensate their respective the 21st cotranslationally inserted amino acid (6), is not suitable, negative impacts on the suppression efficiency. because many organisms need this amino acid for viability and because its UGA-directed insertion requires additional RNA and protein components. Pyl, the 22nd cotranslationally inserted Author contributions: A.A., S.G., and D.S. designed research; A.A., S.G., S.H., C.P., and C.F. amino acid, appears more suited for this purpose, because it is performed research; A.A., S.G., S.H., C.P., and D.S. analyzed data; and A.A. and D.S. wrote the paper. restricted to a small number of organisms, where it accomplishes a special function (7). The Methanosarcinaceae contain a devoted The authors declare no conflict of interest. UAG-recognizing suppressor tRNAPyl (8) and a pyrrolysyl- Abbreviations: Pyl, pyrrolysine; PylRS, pyrrolysyl-tRNA synthetase; DHFR, dihydrofolate reductase. tRNA synthetase (PylRS) dedicated to forming Pyl-tRNAPyl (9, † Pyl Present address: Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, 10). Initial studies indicated that Lys-tRNA can be recognized CEP 21941-901, Rio de Janeiro, Brazil. by bacterial EF-Tu (11) and that in Escherichia coli tRNAPyl acts ‡Present address: Institute for Pharmacy and Molecular Biotechnology, University of Hei- like an amber suppressor (10, 12). delberg, D-69117 Heidelberg, Germany. Pyl A thorough investigation in E. coli of archaeal tRNA should ¶To whom correspondence should be addressed at: Department of Molecular Biophysics Pyl uncover the structural determinants that may make tRNA and and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, PylRS an ideal orthogonal pair when used in bacterial protein CT 06520-8114. E-mail: [email protected]. BIOCHEMISTRY synthesis. Here we investigate the interaction of Methanosarcina © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611634104 PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 ͉ 3141–3146 Downloaded by guest on October 2, 2021 Table 1. Suppression efficiency of M. barkeri tRNAPyl variants (14). However, the U54A mutation resulted only in a minor Mutations Suppression efficiency, % reduction of suppression efficiency, suggesting that the integrity of the T-loop interaction is not critical for tRNAPyl activity and Wild type that A58 could be directly involved in PylRS binding and Fusaro 100 recognition. This idea is supported by the fact that mutation of MS (C44U/A3G) 95 A58 in Desulfitobacterium hafniense tRNAPyl resulted in a Discriminator base Ͼ1,000-fold loss of in vitro aminoacylation efficiency (15). G733C88Base pair mutations G10:C25 to U:A, C13:G22 to A:U in the G733A33D-arm, and G51:C63 to U:A in the T-arm caused significant G733U43reduction of suppression efficiency. Although the observed Acceptor stem effect of mutation of G10:C25 can be attributed to a role in G13A26maintaining the core structure of tRNAPyl via possible interac- C723U30tions with a nucleotide in the variable loop, base pairs C13:G22 G1:C723C:G 70 and G51:C63 do not have obvious structural roles (14), suggest- G2:C713A:U 52 ing a direct contribution of these nucleotides to tRNAPyl fitness A33G58in the translation machinery. A5:U683C:G 100 D-stem and loop Variable Loop. The short variable loop (only three nucleotides U153A39instead of the normal five) is one of the distinctive features of G183U87tRNAPyl. Mutation of any of these nucleotides resulted in strong U203A43reduction of suppression efficiency; the most dramatic effects A213U95were observed in mutants A45G and G48U, and when an G10:C253U:A 50 additional A was inserted to make a 4-nt variable loop. Such A11:U243C:G 88 dramatic effects are consistent with the function of the variable U12:A233G:C 112 loop nucleotides in ensuring the proper relative positioning of C13:G223A:U 25 the two stacked helices that make up the tRNA L-shape. The Insert at G18 47 effect of the A45G mutation may also be considered in light of T␺C-stem and loop the reduction observed upon mutating the first base pair of the U543A80D-arm (G10:C25) because they make a potential tertiary inter- A563U33action (14). A583U40 U603A 108 Anticodon Stem and Loop. The elongated anticodon stem is C50:G643A:U 79 another striking feature of tRNAPyl. Although the mutations of G51:C633U:A 29 the anticodon stem base pairs resulted in only moderate sup- G52:C623U:A 63 pression loss, three mutations are nevertheless worth noticing. G53:C613U:A 63 Disruption of the first base pair of the anticodon stem (A27:U43 Variable loop to G:A) lowered the suppression efficiency. Conversion of the C443U56wobble pair U29a:G41b to a C:G pair or of A31:U39 to C31:G39 A453G23also caused significant reductions of tRNAPyl fitness as a UAG G483U17suppressor. Deletion of the U29a:G41b pair, resulting in a Insert A at C44 17 canonical 5-bp anticodon stem was particularly detrimental to Anticodon stem/loop suppression efficiency.