Q.40 Protein–protein interactions are studied by
P. DNA foot printing
Q. Yeast two hybrid system
R. Ligase chain reaction
S. Mass spectrometry
Correct Answer: (B) Q and S only
Yeast two-hybrid system detects protein-protein interactions via transcriptional activation in yeast, while mass spectrometry identifies interactomes through co-purification and proteomic analysis. DNA footprinting studies DNA-protein binding, and ligase chain reaction amplifies DNA targets.
Option Analysis
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(A) P and S only: Incorrect. DNA footprinting protects DNA from nuclease digestion to map transcription factor binding sites, not protein-protein contacts.
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(B) Q and S only: Correct. Yeast two-hybrid (Q) fuses bait protein to DNA-binding domain and prey to activation domain; interaction reconstitutes transcription. Mass spectrometry (S) via TAP-MS co-purifies complexes for identification.
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(C) P and R only: Incorrect. Ligase chain reaction (LCR) joins adjacent DNA probes for mutation detection/amplification, unrelated to protein interactions.
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(D) Q and R only: Incorrect. LCR detects DNA polymorphisms, not protein-protein interfaces.
Protein-protein interactions detection via yeast two hybrid system and mass spectrometry represents core molecular biology techniques tested in Q.40. Biochemical engineering students must distinguish direct PPI methods from DNA-centric approaches for accurate exam performance.
PPI Detection Methods
Yeast Two-Hybrid (Y2H): Bait (DNA-binding domain fusion) and prey (activation domain fusion) proteins interact to drive HIS3/LacZ reporter expression in SD-Leu-Trp-His media. False positives minimized via mating assays and library screening.
Mass Spectrometry (TAP-MS): Dual affinity purification (Protein A-TEV-Protein C tag) isolates native complexes, followed by LC-MS/MS peptide identification. Quantitative SILAC variants measure interaction stoichiometry.
Non-PPI Methods: DNA footprinting uses DNase I titration to reveal protein-occupied DNA regions (50-200 bp protection). Ligase chain reaction amplifies DNA via thermostable ligase joining hemiphosphorylated probes.
Biotechnology Applications
Y2H screens synthetic biology pathways; MS maps signaling interactomes for kinase inhibitor design. These complement prior Q.35-39 topics (glycolytic localization, RNAi, allosteric kinetics), forming comprehensive molecular toolkit for bioreactor optimization and metabolic engineering.
Exam Strategy
Eliminate DNA techniques (P,R) first; recognize Y2H/MS as gold standards. Quantitative PPI yields interaction confidence scores essential for network modeling in microbial growth kinetics.
