Import of amber and ochre suppressor tRNAs into mammalian cells: a general approach to site-specific insertion of amino acid analogues into proteins.

A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl-tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl-tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.

Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria.

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1-binding sites on 23S rRNA and mRNA.

In Bacteria and Archaea (formerly Archaebacteria) ribosomal protein L1 has a dual function, as a primary rRNA-binding protein and as a translational repressor which binds to its own mRNA. The L1-binding site on the mRNA exhibits high similarity in both sequence and secondary structure to the binding site for L1 on the 23 S rRNA. A sensitive membrane-filter-binding assay has been used to examine the interactions between ribosomal L1 proteins from different archaeal and bacterial species, and 23S rRNA and mRNA fragments from Methanococcus vannielii containing the MvaL1-binding site. Under standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 500 mM KCl), the apparent dissociation constant Kd of the homologous MvaL1-23S rRNA complex is 5 nM, the apparent dissociation constant Kd of the MvaL1-mRNA complex is 0.15 degrees M. L1 proteins from Escherichia coli (EcoL1) and from the thermophilic Bacterium Thermus thermophilus (TthL1), and from the thermophilic Archaea Methanococcus thermolithotrophicus (MthL1), Methanococcus jannaschii (MjaL1), and Sulfolobus solfataricus (SsoL1) were tested for their affinity to the specific L1-binding sites on the 23 S rRNA and mRNA. In general, the affinity of L1 proteins from thermophilic species to the binding sites on both 23 S rRNA and mRNA is about one order of magnitude higher than that of their mesophilic counterparts. This stronger protein-RNA interaction might make a substantial contribution to the thermal tolerance of ribosomes in thermophilic organisms.

Crystals of ribosomal protein L1 from a hyperthermophilic archaeon Methanococcus jannaschii.

Crystallographic studies of ribosomal proteins from bacteria progressed rapidly during the last decade, though the structures of ribosomal proteins from other kingdoms have not yet been published. Here we describe crystals of archaeal ribosomal protein L1 from Methanococcus jannaschii. The protein crystals were grown in 10% PEG 10 K, 50 mM Hepes-HCl (pH 7.5) in hanging drops equilibrated against 33% PEG 10 K, 100 mM Hepes-HCl (pH 7.5). The crystals diffract to at least 2.5 A resolution and belong to the space group P1 with cell parameters a = 34.09 A, b = 39.39 A, c = 55.84 A, alpha = 83.65 degrees, beta = 80.38 degrees, gamma = 75.37 degrees.

MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond.

The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail. As in Escherichia coil, regulation takes place at the level of translation. MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E. coli, was shown to be an autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome. Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies. In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co-regulatory function of MvaL10, the homologue of the regulatory protein L10 of the beta-operon of E. coil. However, we show that MvaL10 does not have a regulatory function.

Use of T7 RNA polymerase in an optimized Escherichia coli coupled in vitro transcription-translation system. Application in regulatory studies and expression of long transcription units.

An Escherichia coli coupled in vitro transcription-translation system has been modified to allow efficient expression of genes under the control of a T7 promoter. We describe both the characterization and use of two S30 crude extracts prepared from E. coli, namely S30 BL21(DE3) (containing endogenous T7 RNA polymerase) and S30 BL21 (supplemented with exogenous T7 RNA polymerase). Since transcription by the highly active T7 RNA polymerase is known to overload the translational machinery of E. coli, the ratio between mRNA and ribosomes has to be regulated in the coupled in vitro system. For this purpose, the level of mRNA is controlled by varying the amount of DNA template (S30 extract with endogenous T7 RNA polymerase) or by limited amounts of exogenously added T7 RNA polymerase. The coupled in vitro system described in this paper provides two especially useful applications. First, it is most suitable for studying the regulation of gene expression in vitro, second, it can be used to express DNA templates carrying up to 10 genes. We show that genes which are not well expressed in E. coli in vivo because of unfavourable codon usage or plasmid instability are synthesized efficiently in the coupled in vitro system.

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.