Lecture 5. Orthogonal Genetic Systems Flashcards
What was the earliest example of mRNA selection?
The ‘specialised ribosome system’ in which modified 30S ribosomal subunits were engineered that recognised correspondingly modified mRNAs in E.coli
What does the bacterial system rely on to locate the transcriptional start site?
Recognition of transcriptional start site in a bacterial system relies on the binding of anti-Shine-Dalgarno regions in ribosomal RNA and Shine-Dalgano regions in the mRNA
What is the role of streptomycin in the specialised ribosome system?
Addition of spectinomycin to cells harbouring both normal and specialised 30S subunits then suppressed translation of non- mutated mRNAs
What is the role of the dual selection process?
The dual selection process is used to identify mRNAlib sequences that do not support translation by wt endogenous ribosomes but which do support translation by ribosomes that contain selected rRNAlib 16S rRNA sequence variants.
What happens in the positive selection part of the dual selection process?
Chloramphenicol acetyltransferase (cat) makes 3-acetoxychloramphenicol which no longer binds to ribo 30S, so no longer toxic to the cell, cells resistant to chloramphenicol providing protection to the cell
What happens in the negative selection part of the dual selection process?
Uracil phosphoribosyltransferase converts uracil to UMP and (added) 5-FU to 5-fluoro-dUMP which inhibits thymidylate synthase and is toxic to the cell
What happens to cells with strong and weak pairings in the dual selection process?
Cells with a strong pairing will survive chloramphenicol but die to 5-FU
Other way round for cells with weak parings
What did the dual selection process allow scientists to do?
Very thorough system allowed stringent selection to libraries, gave rise to more than a billion different combinations of ribosomal RNA and mRNA sequences
What must be done to investigate the whole ribosome?
In order to investigate the whole ribosome, you need to make mutations in the 30S and 50S and tether them together to create the so-called Ribo-T
What is the use of orthogonal rRNA-mRNA pairs?
Can place the synthesis of the 16S ribosomal RNA gene under the control of an inducible promoter so that you can switch it on whenever you like, enabling the cells to make modified ribosomes that recognise the mutant mRNA and will take over the whole activity of protein synthesis machinery
Turning on and off ensures cell doesn’t overburn due to hijacking
What are orthogonal rRNA-mRNA pairs?
Orthogonally translated subsets of mRNAs systems have been engineered in E.coli by modifying rRNA sequences and mRNA 5’UTRs, thus creating paired ribosome-mRNA populations that can be regulated independently of the main transcriptome.
What happens in reassignment of the genetic code?
Reassignment involves changes to tRNA anticodons, wobble nucleotide modifications, aminoacyl-tRNA synthetase recognition of cognate tRNAs, release factor binding of stop codons. There are likely to be more sense- and nonsense- codon reassignments that have yet to be characterised
What are the 21st and 22nd amino acids?
Selenocysteine and Pyrrolysine (Pyl)
How can selenocysteien be coded?
Can be coded by UGA if the mRNA includes a selenocysteine insertion sequence (SECIS); Incorporation also requires a special type of seryl-tRNA (tRNASec) and a unique translation elongation factor (SELB in prokaryotes; eEFSec + SECIS Binding Protein 2 in eukaryotes)
How can pyrrolysine be incorportated?
incorporation (in methanogens) proceeds via a specific tRNA called tRNAPyl, whereby this species is charged by pyrrolysyl-tRNA synthetase. Incorporation occurs at UAG, in competition with the binding of release factors and, as far as we know, does not require a special elongation factor or mRNA element
What is the minimum number of codons that E. coli can have?
57/64, means more codons available for reassignment
What are quadruplet codons?
Codons with 4 bases (large number of additional codon options)
Give an example of a molecule that could incorporate a novel amino acid?
Myoglobin and AGGA (read as L-homoglutamine)
How can chemistry be used to create recording diversity in vitro?
Combined mutations in the 23S rRNA with flexible tRNA acylation enzymes (flexizymes, substituting for aminoacyl transferases) to create an orthogonal reassignment system.
What does the flexizyme recognise in the orthogonal reassignment system?
The aromatic ring (activated by the CME group)
For a non-aromatic amino acid, dinitro benzyl ester (DBE) is recognised by the flexizyme
What conclusions did Ribo-T allow scientists to reach?
Ribo-T ribosomes are functional, thus demonstrating that ribosome assembly does not have to proceed via separate subunits (covalently bound, cannot be seperated)
What are xeno nucleic acids (XNA)?
Alternative structures that are not found
What position of ribose is most prone to change?
2’ position (traditionally a -OH group)
Which of the four pentoses (ribose, arabinose, xylose and lyxose) is the only one that allows for free rotation of the functional groups?
Only β-D-ribose allows free rotation of the functional groups OH, phosphate and the base (G, A, T(U), C) and supports formation of double- stranded structures.
What are the applications of aptamers?
Aptamers are used in a diversity of applications, including as therapeutic drugs, biosensors (pathogen
recognition, cancer recognition, monitoring environmental contamination etc.)
What aptamers are used today?
Partially modified RNA and DNA aptamers and, more recently, fully XNA aptamers have
been developed, with the objective of improving aptamer properties such as biostability and
functionality
