Bacterial Genome Containing Chimeric DNA-RNA Sequences (original) (raw)
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Replacement of 2'-Deoxycytidine by 2'-Deoxycytidine Analogues in the E. coli Genome
Journal of the American Chemical Society, 2016
Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.
Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes
Journal of bacteriology, 1990
The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.
A new RNA synthesis mutant of E. coli
Biochemical Genetics, 1971
A temperature-sensitive mutant ofE. coli is described. At the nonpermissive temperature, the capacity for RNA and protein synthesis decreases logarithmically in the mutant. The mutant is unable to support the growth off2 or T7 virus, even at the permissive temperature. The temperature-sensitive mutation maps approximately 1' away from rifr in E. coli and therefore affects a gene previously undescribed. The temperature sensitivity is suppressed by sublethal concentrations of rifampicin. Moreover, in rifrT s double mutants, the T s mutation suppresses rifr and vice versa. The partially purified RNA polymerases from mutant and wild-type cells have different temperature and salt optima.
1998
Escherichia coli rRNA contains 10 pseudouridines of unknown function. They are made by synthases, each of which is specific for one or more pseudouridines. Here we show that the sfhB (yfiI) ORF of E. coli is a pseudouridine synthase gene by cloning, protein overexpression, and reaction in vitro with rRNA transcripts. Gene disruption by miniTn10(cam) insertion revealed that this synthase gene, here renamed rluD, codes for a synthase which is solely responsible in vivo for synthesis of the three pseudouridines clustered in a stem-loop at positions 1911, 1915, and 1917 of 23S RNA. The absence of RluD results in severe growth inhibition. Both the absence of pseudouridine and the growth defect could be reversed by insertion of a plasmid carrying the rluD gene into the mutant cell, clearly linking both effects to the absence of RluD. This is the first report of a major physiological defect due to the deletion of any pseudouridine synthase. Growth inhibition may be due to the lack of one or more of the 23S RNA pseudouridines made by this synthase since pseudouridines 1915 and 1917 are universally conserved and are located in proximity to the decoding center of the ribosome where they could be involved in modulating codon recognition.
Journal of Molecular Biology, 2011
Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA-but a single modified nucleoside at position 2501 in 23S rRNA-has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine-a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.
Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli
Biochimie, 1991
The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth. Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules. We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression. In the first part of the study the function of AT-rich sequences upstream of the-35 hexamer of rRNA promoter PI in the activation of rRNA transcription was analyzed. We confirm that a sequence dependent bend upstream of PI is responsible for the high promoter activity. Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation. In addition, the efl~ct of the trans activating protein Fis on the transcription initiation of promoter PI was investigated. We can show, using the abortive initiation assay, that the predominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant KB) while the isomerisation rate (/:f) from a closed to an open RNA polymerase promoter complex is not altered significantly. We also describe the characterizatior~ of sequence determinants impoltant for stringent regulation and growth rate control. Evidence is provided that the discriminator wotif GCGC is a necessary but not sufficient element for both types of control. Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp). Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA. We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions. We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.
Expanding the Genetic Code of Escherichia coli
Science, 2001
A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.
Biochemistry, 1995
Pseudouridine (Y) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one Y, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-36841. To assess the function of this single Y residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 23 1 amino acids and has a calculated molecular mass of 25 836 Da. It converts U516 in E. coli 16s RNA transcripts into Y but does not modify any other position in this RNA. It does not react with free unmodified 16s RNA at all, and only poorly with 30s particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30s ribosomal proteins. The yield varied from 0.6 to 1 .O mol of Y/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23s RNA and tRNAVa' transcripts were also inactive. These results suggest that Y formation in vivo occurs at an intermediate stage of 30s assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene.
Cell, 1976
Transducing phage hrifdll carries an rRNA transcription unit containing genes for 5S, 18S, and 235 rRNAs and also tRNA:lu. Mutants were isolated from this phage that carry deletions removing various amounts of the distal end of this transcription unit. These deletions were physically mapped on the Xrifdl8 phage genome. Synthesis of rRNAs and of tRNAglu was examined in ultravioletirradiated E. coli cells infected with hrifdl8 or with various deletion mutants. It was observed that mutant phages in which the distal end (the 5s rRNA gene and a part of the 235 rRNA gene) of the rRNA transcription unit is deleted can still synthesize both 18s rRNA (or its precursor) and tRNA:lu. Apparently, the post-transcriptional cleavage that produces these RNA molecules does not require the presence of the entire transcription unit, that is, it can take place without the complete structure of the transcript ("30s pre-ribosomal RNA"). In addition, the experimental results support the gene order, 16s rRNA, tRNA$+lu, 23s rRNA, and 5s rRNA genes, in the rRNA transcription unit carried by hrifdl8.
Large scale expression and purification of recombinant RNA in Escherichia coli
Methods, 2011
Stable, folded RNA are involved in many key cellular processes and can be used as tools for biological, pharmacological and/or molecular design studies. However, their widespread use has been somewhat limited by their fragile nature and by the difficulties associated with their production on a large scale, which were limited to in vitro methods. This work reviews the novel techniques recently developed that allow efficient expression of recombinant RNA in vivo in Escherichia coli. Based on the extensive data available on the genetic and metabolic mechanisms of this model organism, conditions for optimal production can be derived. Combined with a large repertoire of RNA motifs which can be assembled by recombinant DNA techniques, this opens the way to the modular design of RNA molecules with novel properties.