ABSTRACT
In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNA(cys)(Amber) from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNA(cys)(Amber) with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNA(cys)(Amber), which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Bacillus subtilis/genetics , Escherichia coli/genetics , RNA, Transfer, Cys/biosynthesis , RNA, Transfer, Cys/chemistry , Amino Acyl-tRNA Synthetases/genetics , Cell-Free System , Escherichia coli/metabolism , Fluorescent Dyes/chemistry , In Vitro Techniques , Models, Molecular , Protein Biosynthesis , RNA Stability , RNA, Bacterial/biosynthesis , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Transfer, Cys/genetics , Transfer RNA AminoacylationABSTRACT
Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.
Subject(s)
Amino Acyl-tRNA Synthetases/analysis , Electrophoresis, Polyacrylamide Gel/methods , RNA, Transfer/analysis , Animals , Archaea/metabolism , Blotting, Northern/methods , Humans , Hydrogen-Ion Concentration , Lysine/analogs & derivatives , Lysine/biosynthesis , Protein Engineering/methods , Pyrimidine Nucleosides/biosynthesis , RNA, Bacterial/isolation & purification , RNA, Transfer/isolation & purification , RNA, Transfer, Cys/biosynthesis , RNA, Transfer, Ile/metabolism , RNA, Transfer, Met/metabolism , UreaABSTRACT
In nature, ribosomally synthesized proteins can contain at least 22 different amino acids: the 20 common amino acids as well as selenocysteine and pyrrolysine. Each of these amino acids is inserted into proteins codon-specifically via an aminoacyl-transfer RNA (aa-tRNA). In most cases, these aa-tRNAs are biosynthesized directly by a set of highly specific and accurate aminoacyl-tRNA synthetases (aaRSs). However, in some cases aaRSs with relaxed or novel substrate specificities cooperate with other enzymes to generate specific canonical and non-canonical aminoacyl-tRNAs.
Subject(s)
Transfer RNA Aminoacylation , Amino Acyl-tRNA Synthetases/metabolism , Aspartate-tRNA Ligase/metabolism , Bacteria/enzymology , RNA, Transfer, Amino Acyl/biosynthesis , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/metabolism , RNA, Transfer, Asn/biosynthesis , RNA, Transfer, Asn/chemistry , RNA, Transfer, Cys/biosynthesis , RNA, Transfer, Cys/chemistry , RNA, Transfer, Gln/biosynthesis , RNA, Transfer, Gln/chemistryABSTRACT
A number of archaeal organisms generate Cys-tRNA(Cys) in a two-step pathway, first charging phosphoserine (Sep) onto tRNA(Cys) and subsequently converting it to Cys-tRNA(Cys). We have determined, at 3.2-A resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, alpha(4) synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (alphabeta)(2) phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Methanococcus/chemistry , Methanococcus/enzymology , Phosphoserine/metabolism , RNA, Transfer, Cys/biosynthesis , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Diphosphates/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Structural Homology, Protein , Thermus thermophilus/enzymologyABSTRACT
We have isolated and sequenced chloroplast (chl) and cytoplasmic (cyt) cysteine tRNAs from Nicotiana rustica. Both tRNAs carry a GCA anticodon but beyond that differ considerably in their nucleotide sequences. One obvious distinction resides in the presence of N6-isopentenyladenosine (i6A) and 1-methylguanosine (m1G) at position 37 in chl and cyt tRNA(Cys) respectively. In order to study the potential suppressor activity of tRNAs(Cys) we used in vitro synthesized zein mRNA transcripts in which an internal UGA stop codon had been placed in either the tobacco rattle virus (TRV)- or tobacco mosaic virus (TMV)-specific codon context. In vitro translation was carried out in a messenger- and tRNA-dependent wheat germ extract. Both tRNA(Cys) isoacceptors stimulate read-through over the UGA stop codon, however, chl tRNA(GCA)Cys is more efficient than the cytoplasmic counterpart. The UGA in the two viral codon contexts is suppressed to about the same extent by either of the two tRNAs(Cys), whereas UGA in the beta-globin context is not recognized at all. The interaction of tRNA(GCA)Cys with UGA requires an unconventional G:A base pair in the wobble position, as postulated earlier for plant tRNA(G psi A)Tyr misreading the UAA stop codon. This is the first case that a cysteine-accepting tRNA has been characterized as a natural UGA suppressor.
