Hello,
I am trying to knockout a bacterial gene X which is overlapping with a tRNA gene. This overlapping tRNA gene has only one copy in genome So seems to be essential. The target gene X and the overlapping tRNA genes are on opposite DNA strands and they are transcribed in opposite direction. Before knocking out the target gene X, I am thinking of either inserting a copy of this overlapping tRNA gene in place of any known non-essential gene in genome of bacteria by homologous recombination or episomally express the tRNA gene in the bacteria. I have few queries regarding cloning and expression of tRNA gene.
1) If I express tRNA episomally, Does it require to include a promoter at 5 prime end of the tRNA in the vector. Because I read that tRNA genes have internal promoters.
2) Should it be beneficial to clone up to 80-150 bp 5 prime upstream and 3 prime downstream sequences of tRNA genes so that it may require sequences for its maturation.
Hi Ajay Kumar
follow this articale that mentioned
Analysis of 100 complete sets of the cytoplasmic elongator tRNA genes from Bacteria, Archaea, and Eukarya pointed to correspondences between types of anticodon and composition of the rest of the tRNA body. The number of the hydrogen bonds formed between the complementary nucleotides in the anticodon–codon duplex appeared as a major quantitative parameter determining covariations in all three domains of life. Our analysis has supported and advanced the “extended anticodon” concept that is based on the argument that the decoding performance of the anticodon is enhanced by selection of a matching anticodon stem–loop sequence, as reported by Yarus in 1982. In addition to the anticodon stem–loop, we have found covariations between the anticodon nucleotides and the composition of the distant regions of their respective tRNAs that include dihydrouridine (D) and thymidyl (T) stem–loops. The majority of the covariable tRNA positions were found at the regions with the increased dynamic potential—such as stem–loop and stem–stem junctions. The consistent occurrences of the covariations on the multigenomic level suggest that the number and pattern of the hydrogen bonds in the anticodon–codon duplex constitute a major factor in the course of translation that is reflected in the fine-tuning of the tRNA composition and structure.
Article Analysis of genomic tRNA sets from Bacteria, Archaea, and Eu...
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