Expansion of the Genetic Code Through the Use of Modified Bacterial Ribosomes

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, β-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.     

Ribosome pausing and stacking during translation of a eukaryotic mRNA.

We have devised a sensitive assay to determine the distribution of translating ribosomes on a mRNA. Using this assay to monitor ribosome transit on bovine preprolactin mRNA, we have detected four major positions of ribosome pausing in both wheat-germ and rabbit reticulocyte extracts. Two of these rate-limiting steps represent initiation and termination. One pause occurs after approximately 75 amino acids have been polymerized; signal recognition particle arrests preprolactin synthesis at this position. The other internal pause occurs at 160 amino acids. In these latter two cases ribosomes stop at a GGC glycine codon; however, two other GGC codons are translated without apparent pausing. Surprisingly, we find that up to nine ribosomes are tightly stacked behind each pausing ribosome, such that the ribosome centers are only 27-29 nucleotides apart. The assay should prove useful for probing mechanisms of translational regulation.     

Cis control of gene expression in E.coli by ribosome queuing at an inefficient translational stop signal

An UGA stop codon context which is inefficient because of the 3'-flanking context and the last two amino acids in the gene protein product has a negative effect on gene expression, as shown using a model protein A' gene. This is particularly true at low mRNA levels, corresponding to a high intracellular ribosome/mRNA ratio. The negative effect is smaller if this ratio is decreased, or if the distance between the initiation and termination signals is increased. The results suggest that an inefficient termination codon can cause ribosomal pausing and queuing along the upstream mRNA region, thus blocking translation initiation of short genes. This cis control effect is dependent on the stop codon context, including the C-terminal amino acids in the gene product, the translation initiation signal strength, the ribosome/mRNA ratio and the size of the mRNA coding region. A large proportion of poorly expressed natural Escherichia coli genes are small, and the weak termination codon UGA is under-represented in small, highly expressed E.coli genes as compared with the efficient stop codon UAA.     

Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.

The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X-ray diffraction methods. This primary RNA-binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha-helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha-helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha-helix has an architectural role and serves to fix the relative separation and orientation of the N- and C-terminal domains within the ribosome. The N-terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM).     

Differential rate of ribosomal protein synthesis in Escherichia coli B-r

The differential rate of ribosomal protein synthesis, α_r (ribosomal protein synthesis rate/total protein synthesis rate), was measured for Escherichia coli strain B/r growing at different steady-state rates ranging from 0.67 to 2.3 doublings/hour. For growth rates above 1.2 doublings/hour, α_r was found to be proportional to the growth rate μ (doublings/h), such that α_r = 0.09 μ, and the ribosome efficiency (amino acids polymerized/second per ribosome), calculated from α_r, was found to be 14 to 18 amino acids/second per ribosome. With decreasing growth rates below 1.2 doublings/hour, α_r was found to be increasingly greater than 0.09 μ and the ribosome efficiency gradually decreased such that at μ = 0.67, α_r = 0.085, and the ribosome efficiency was reduced by 30% and was equal to 10 to 13 ammo acids/second per ribosome. These results imply that the protein to DNA ratio is constant for μ > 1.2 and equal to 4 × 10⁸ to 5 × 10⁸ amino acids/genome. For μ < 1.2, this ratio gradually decreases such that at μ = 0.67, protein to DNA = 3 × 10⁸ to 4 × 10⁸ amino acids/genome. These relationships were verified by direct measurements of the amounts of DNA, RNA and protein at different steady-state growth rates. In addition, protein accumulation was measured following a nutritional shift-up from succinate to glucose minimal medium. The results indicate that the ribosome efficiency increases by approximately 40% within the first few minutes following the shift-up.     

Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.     

Primary structure of the DNA terminal protein of bacteriophage PRD1.

The genome of a lipid-containing phage, PRD1, is replicated by a protein-priming mechanism. We have determined the nucleotide sequence of the PRD1 gene 8 which specifies the terminal protein, the protein primer for DNA synthesis. The coding region is 780 base pairs long and encodes for 259 amino acids (29,326 daltons). The predicted amino acid sequence of the PRD1 terminal protein reveals no substantial homology with that of any known terminal protein. However, hydropathy profiles of the PRD1, phi 29, and Nf terminal proteins are remarkably similar, suggesting a common evolutionary origin. A particular tyrosine residue is predicted to be covalently linked to the 5' end of the PRD1 DNA. The initiation codon ATG of gene 8 is preceded by the identifiable ribosome binding site, and putative promoter sequences. There are unique palindromic sequences between the ribosome binding site and "-10" region.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.     

Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography

Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 Å resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer-template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket.     

原噬菌体PBSX阻遏基因及其温度敏感型等位基因的克隆分析

本研究利用聚合酶链式反应技术,成功地克隆了枯草芽孢杆菌缺陷型原噬菌体ＰＢＳＸ阻遏基因及其温度敏感型等位基因。核苷酸序列分析发现,野生型及其温度敏感型阻遏基因之间的碱基变异较大,但却存在几乎完全相同的开放读框,尤其是开放读框ｏｒｆⅠ,可能编码着１１３个氨基酸的阻遏蛋白,并且还推定了开放读框的启动区和核糖体结合位点。通过互补实验,证实了野生型阻遏基因的产物能够抑制温度诱导ＰＢＳＸ原噬菌体,表明克隆的基因有着正常的生物活性。     

A hypothesis and an experiment on the origin of the genetic coding process

From thermodynamic considerations it is argued that the earliest templates for protein synthesis were very unlike present nucleic acids. It is suggested that they were composed of linked uracil derivatives bearing hypothetical side chains that could specifically recognize and interact with individual amino acid side chains (Fig. 1). An experiment supporting the possibility of such a specific interaction is described. The hypothesis accounts for the grouping of amino acids in the present codon table according to their relative hydrophobic character.     

Characterization of the Trypanosoma cruzi ortholog of the SBDS protein reveals an intrinsically disordered extended C-terminal region showing RNA-interacting activity

The human SBDS gene and its yeast ortholog SDO1 encode essential proteins that are involved in ribosome biosynthesis. SDO1 has been implicated in recycling of the ribosomal biogenesis factor Tif6p from pre-66S particles as well as in translation activation of 60S ribosomes. The SBDS protein is highly conserved, containing approximately 250 amino acid residues in animals, fungi and Archaea, while SBDS orthologs of plants and a group of protists contain an extended C-terminal region. In this work, we describe the characterization of the Trypanosoma cruzi SBDS ortholog (TcSBDS). TcSBDS co-fractionates with polysomes in sucrose density gradients, which is consistent with a role in ribosome biosynthesis. We show that TcSBDS contains a C-terminal extension of 200 amino acids that displays the features of intrinsically disordered proteins as determined by proteolytic, circular dichroism and NMR analyses. Interestingly, the C-terminal extension is responsible for TcSBDS–RNA interaction activity in electrophoretic mobility shift assays. This finding suggests that Trypanosomatidae and possibly also other organisms containing SBDS with extended C-terminal regions have evolved an additional function for SBDS in ribosome biogenesis.     

Amino acid receptor sites in the facial taste system of the sea catfish Arius felis

1. The amino acid sensitivity and specificity of the facial taste system of the marine catfish, Arius felis, is characterized electrophysiologically.
2. The facial taste system of Arius felis responded to all 28 amino acids tested, but was highly sensitive to only a few. In general, acidic amino acids and neutral amino acids with short side chains were more effective than imino, basic and neutral amino acids with long side chains.
3. A reciprocal cross-adaptation protocol used to characterize the receptor sites identified at least some relatively independent receptor sites for L-arginine, L-histidine, L-proline, L-alanine, glycine, D-alanine and L-glutamate.
4. Of the 7 amino acids that were indicated to have relatively independent receptor sites, the median electrophysiological threshold for L-alanine, the most stimulatory, and L-proline, the least stimulatory compounds, were 10 nM and 10,000 nM, respectively. The integrated facial taste response did not saturate at test amino acid concentrations up to 10 mM.
5. The generalized depression in responsiveness to test stimuli observed during amino acid adaptation is proposed to be a result of the co-distribution of sensitivity at the level of single taste cells rather than high cross-reactivity of the respective amino acid receptor sites for the test stimuli.

     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

Pure translation display

Methods such as monoclonal antibody technology, phage display, and ribosome display provide genetic routes to the selection of proteins and peptides with desired properties. However, extension to polymers of unnatural amino acids is problematic because the translation step is always performed in vivo or in crude extracts in the face of competition from natural amino acids. Here, we address this restriction using a pure translation system in which aminoacyl-tRNA synthetases and other competitors are deliberately omitted. First, we show that such a simplified system can synthesize long polypeptides. Second, we demonstrate "pure translation display" by selecting from an mRNA library only those mRNAs that encode a selectable unnatural amino acid upstream of a peptide spacer sequence long enough to span the ribosome tunnel. Pure translation display should enable the directed evolution of peptide analogs with desirable catalytic or pharmacological properties.     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.