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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
Published on: August 21, 2018
Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species
C Yarian1, M Marszalek, E Sochacka
1Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Biochemistry
|November 7, 2000
Summary
Modified nucleosides in tRNA anticodon loops are crucial for ribosome binding. Specific modifications like t(6)A and mnm(5)U in tRNA(Lys) restore binding to poly-A programmed ribosomes and are essential for AAG programmed ribosomes.
Area of Science:
- Molecular Biology
- RNA Biochemistry
Background:
- Nucleoside modifications are essential for tRNA structure and function.
- Human tRNA(Lys3)(UUU) anticodon stem-loop (ASL) is unable to bind poly-A programmed ribosomes.
Purpose of the Study:
- To investigate the role of specific modified nucleosides in the tRNA(Lys) anticodon loop for ribosomal binding.
- To determine if these modifications affect thermal stability.
Main Methods:
- Chemical synthesis of ASL(Lys) heptadecamers with site-specific incorporation of modified nucleosides: pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)), and N6-threonylcarbamoyl-adenosine (t(6)A(37)).
- Assessed ribosomal binding ability of modified ASL constructs to poly-A and AAG programmed ribosomes.
Main Results:
- Incorporation of t(6)A(37) and mnm(5)U(34) enhanced ASL(Lys3)(UUU) structure and significantly improved binding to poly-A programmed ribosomes.
- Both t(6)A(37) and mnm(5)U(34) were required for ASL(Lys3)(UUU) to bind AAG programmed ribosomes.
- The combination of t(6)A(37) and mnm(5)U(34) increased ASL(Lys3)(UUU) affinity for poly-A programmed ribosomes to levels comparable to native E. coli tRNA(Lys).
- Unmodified human tRNA(Lys1,2)(CUU) ASL bound AAG but did not wobble to AAA.
Conclusions:
- Anticodon loop modifications in tRNA(Lys)(UUU) are necessary to impart ordered structure for ribosomal binding to AAA.
- A combination of modified nucleosides is required for binding to AAG programmed ribosomes.
- Specific nucleoside modifications dictate tRNA's ability to interact with different ribosomal contexts.
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