Cell-free Coupled Transcription-translation System Research Articles

Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar hook.

This paper reports on the organization, expression, and function of the divergently transcribed flbG and flaN operons in the hook gene cluster of Caulobacter crescentus. The transcription initiation site of flbG was determined previously, and in this work the transcription map was completed by locating the 3' end of the mRNA using nuclease S1 protection assays. A previous genetic study had suggested that the flbG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5'-flbG, flbH, and flgE. FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaffolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein. The divergently transcribed flaN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins. Based on the amino acid sequence similarities and phenotypes of mutants, flbG, flbH, and flaN have been renamed fliK, flgD, and flgK, FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identified as part of a 190-kDa FliK-LacZ fusion protein. We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly.

The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related.

Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese

A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.

Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.

For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.

Characterization of a new class of tetracycline-resistance gene tet(S) in Listeria monocytogenes BM4210.

The nucleotide sequence of the tetracycline (Tc)-minocycline (Mc)-resistance determinant of plasmid pIP811 from Listeria monocytogenes BM4210 has been determined. The gene, designated tet(S), was identified by analysis of the start and stop codons as a coding sequence of 1923 bp, corresponding to a protein with a calculated M(r) of 72,912. The apparent 68-kDa size estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the protein characterized in a cell-free coupled transcription-translation system was in good agreement with the calculated value. The tet(S) gene product exhibits 79 and 72% amino acid identity with Tet(M) from Streptococcus pneumoniae and Tet(O) from Campylobacter coli, respectively. The distribution of tet(S) in strains of Gram+ and Gram- genera resistant to Tc (TcR) and Mc (McR) was studied by hybridization under high stringency using a 590-bp intragenic probe. Homology with tet(S) was detected in two clinical isolates of L. monocytogenes isolated in different geographical areas.

Nucleotide sequence of the fosB gene conferring fosfomycin resistance in Staphylococcus epidermidis

We have determined the nucleotide sequence of gene fosB of plasmid pIP1842, which confers resistant to fosfomycin in Staphylococcus epidermidis BM2641. The resistance gene was identified as a coding sequence of 417 bases pairs corresponding to a protein with a calculated Mw of 16,345 daltons. This value is in good agreement with that of 15,000, estimated by SDS-polyacrylamide gel electrophoresis of a bacterial cell-free coupled transcription-translation system. The fosB gene product exhibits 48% sequence homology with the fosfomycin resistance protein FOSA of Enterobacteriaceae. The substitutions are scattered throughout indicating that the two corresponding genes have diverged from a common ancestor. Downstream from fosB is located an open reading frame that was partially sequenced. The deduced amino-acid sequence was 50% homologous to REP alpha 1, a protein involved in the replication of the Enterococcus faecalis plasmid pAM alpha 1.

Molecular characterization of two proteins involved in the excision of the conjugative transposon Tn1545: homologies with other site-specific recombinases.

Excision is probably the initial and rate-limiting step of the movements of conjugative transposons of Gram-positive bacteria such as Tn916 and Tn1545. We have shown, by molecular cloning and DNA sequencing, that a 2058 bp Sau3A right-junction fragment of transposon Tn1545 specifies two gene products that are involved in the excision of the element. The DNA sequence of these genes, designated orf1 and orf2, has been determined and the corresponding proteins, ORF1 and ORF2, have been identified in a bacterial cell-free coupled transcription-translation system. These proteins are freely diffusible since they are able to trans-complement in vivo a deletion derivative of Tn1545 defective for excision. Using an in vivo complementation assay, we have demonstrated that ORF2 alone is able to catalyse excision and that ORF1 strongly stimulates the activity of ORF2. We also found that ORF1 and ORF2 display local homology with, respectively, proteins Xis and Int from lamboid phages, which suggests that these excision systems have a common origin. Based on the functional properties of the integrase of bacteriophage lambda, on the analysis of the nucleotide sequence of the junction fragments and of the target before insertion and after excision, a model is proposed for ORF2-catalysed excision of Tn1545 and related conjugative transposons.

Cloning and expression of the branching enzyme gene ( glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli

Using the glgB gene from Escherichia coli as a hybridization probe, the gene encoding the branching enzyme of the cyanobacterium Synechococcus sp. PCC7942 has been identified on a 3.9-kb PstI fragment which was cloned into plasmid pUC9. Two types of plasmids have been isolated. Plasmid pKVNl was expressing the Synechococcus sp. gene as was shown by complementation of the glgB mutation of E. coli KV832. Plasmid pKVN2, which carried the same insert in the opposite orientation was unable to complement E. coli KV832, indicating that the promoter of the cloned gene was either absent or was not recognized in E. coli. Determination of branching activity in extracts of Synechococcus sp. and E. coli KV832[pKVNl] showed that the enzyme was optimally active at approximately 35°C. No significant activity was present at temperatures higher than 55°C, reflecting the mesophilic nature of the cloned enzyme. In a cell-free coupled transcription-translation system the cloned gene specified two proteins of 84 kDa and 72 kDa, respectively, which are probably translated independently from the same gene by initiation at two different start codons.

Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli

A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants.

Studies in vitro on the flavinylation of 6-hydroxy-D-nicotine oxidase.

The gene of 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell-free coupled transcription-translation system derived from Escherichia coli MZ9. Ultraviolet irradiation of the E. coli extract did not affect synthesis of the 6-HDNO polypeptide nor total protein synthesis but enzymatic 6-HDNO activity could not be detected. Addition of FAD to the irradiated cell extract restored the capability of the transcription-translation assays to synthesize enzymatically active 6-HDNO. However, enzymatic activity could not be restored on addition of FAD plus cell-free extract to the ultraviolet-inactivated assays after completion of apo-6-HDNO synthesis (60 min) nor to immunoprecipitates thereof. Under similar conditions, addition of [14C]FAD did not increase the protein-bound radioactivity. These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo-6-HDNO-polypeptide was synthesized. It was, however, not possible to bind the cofactor to the completed polypeptide chain. These findings argue for a cotranslational cofactor binding.

Localization of the gene for P700 chlorophyll a protein in pea chloroplast DNA

The gene for the P700 chlorophyll a protein (CPI) has been located in pea chloroplast DNA, using a cell-free coupled transcription-translation system from E. coli programmed with total pea chloroplast DNA and cloned restriction fragments of pea chloroplast DNA. Poly-peptides related to CPI were identified by immunoprecipitation with specific antibodies raised against CPI from pea, and protein A-Sepharose. The gene is located between the genes for the α subunit of ATP synthase and cytochrome f.

Localization of the gene for cytochrome f in pea chloroplast DNA

Pea chloroplast DNA and cloned restriction fragments of pea chloroplast DNA have been used as templates in a cell-free coupled transcription-translation system from E. coli and have been shown to direct the synthesis of a 39 kD polypeptide related to cytochrome f (37.3 kD). The product was shown to be similar to cytochrome f by immunoprecipitation with specific antibodies and by peptide mapping of the products of limited chymotrypsin digestion. The structural gene for this higher molecular weight form of cytochrome f has been located within a 3.3 kbp BglII fragment situated 18 kbp from the single 16S rRNA gene.

Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro.

Expression of protein synthesis initiation factor IF-3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage. A strain haploid for infC, the gene for IF-3, was modified to carry one or two additional infC genes giving diploid and triploid strains. Polyploid strains were achieved by the presence of multicopy plasmids expressing the infC gene. When IF-3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF-3, enabling large quantities to be purified. When lysates were prepared from freshly grown cells, only IF-3 alpha (the long form) was detected; however when IF-3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF-3 beta (the short form, lacking six amino acids from the N terminus). The synthesis of the two IF-3 forms was also studied by using a cell-free coupled transcription-translation system dependent on exogenous DNA: the IF-3 gene was found to be very efficiently expressed. IF-3 alpha increased more rapidly than IF-3 beta but following the cessation of protein synthesis IF-3 alpha decreased while IF-3 beta still increased. The results suggest that IF-3 alpha is slowly degraded to the beta form. Addition of non-radioactive IF-3 alpha, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF-3 synthesis. Synthesis of IF-3 in vitro appears to be sensitive to guanosine 3'-diphosphate 5'-diphosphate.

Cell-free coupled transcription-translation system for investigation of linear DNA segments.

Heretofore the DNA-directed coupled transcription-translation system, most useful in gene expression analysis, has been limited to the use of circular or long linear DNAs. Linear DNAs are degraded in this system by an exonucleolytic activity that can be eliminated by making the synthetic extracts from a suitable recB mutant of Escherichia coli. Using these extracts, we have examined the gene expression of a variety of linear DNAs. In particular, the complex pattern of expression of ribosomal protein genes and RNA polymerase genes in the rpoBC-rplLJ region has been analyzed by comparing the protein products obtained when using lambda rifd18 DNA with the product obtained when using the same DNA segmented with various restriction enzymes. The results obtained confirm the conclusions of others obtained by much more elaborate in vivo techniques. It seems highly likely that this cell-free system will have extensive applications in the area of analysis of gene expression.

Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3

A hybrid plasmid, pJC74, carrying the large (3.35 Md) EcoRI- PstI fragment of ColE1, the 1.3 and 1.8 Md PstI fragments of plasmid R1 drd19 (containing a part of Tn3 and known to specify transposition functions), and a part of λ bacteriophage DNA carrying the fused cohesive ends of λ ( cos) was constructed. Supercoiled DNA of pJC74 and a series of deletions (series pJC75) were used in a cell-free coupled transcription-translation system. Analysis of the proteins produced allowed the identification of a 28 to 30 kd protein and/or a 3 kd protein responsible for mobilisation of the plasmids in sex-factor-promoted conjugation. Furthermore, three protein bands of 12, 12.5 and 13 kd were correlated with the presence of a portion of the Tn 3 transposon previously. shown to code for transposition functions which can complement in trans. These latter and the 30 kd β-lactamase were the only proteins identified as Tn 3-specific by comparison of ColE1 with RSF2124.

Synthesis of colicin E1 in a cell-free coupled transcription-translation system

Protein was synthesized in a cell-free coupled transcription-translation (S30) system on plasmid Col E1 and phage T2 and Sd DNA templates. Phage T2 DNA and closed plasmid DNA proved to be the most active templates. The latter controlled the synthesis of eight individual proteinsin vitro, including the biologically active protein antibiotic colicin. The titer of colicin synthesized in this cell-free system reached 1024 units/ml, two orders of magnitude higher than the titer of the antibiotic in populations of colicinogenic bacteria.