Chem5520 Lecture 7a

Overview of Lecture 7

General Information

  • Last lecture before spring break, providing a crucial recap of important concepts.

  • Introduction of a new quiz format on Canvas, details to help students prepare effectively.

Focus Areas

  • The primary focus is on engineering the central dogma of molecular biology, which encompasses DNA, RNA, and protein synthesis.

  • Particular attention is given to the incorporation of non-natural amino acids and the role of nucleic acids in this process.

Chemical Aminoacylation

  • Discussion on the synthesis of aminoacyl tRNA: the process critical for translating genetic information into proteins.

  • Emphasizing the importance of modified tRNA synthetases in integrating non-natural amino acids into proteins, expanding the functional repertoire of proteins.

  • Challenges faced in the incorporation of non-natural amino acids, highlighting their complexity and potential for enhancing protein function in biotechnology.

Earlier Experiments

Seymour Benzer’s 1962 Experiment:

  • An experimental landmark that demonstrated the ribosomes' ability to incorporate non-natural amino acids into polypeptides.

  • The experiment utilized cysteine tRNA along with radioactive sulfur to track the incorporation of synthesized amino acids.

  • Significant finding: The ribosomal operation is contingent on codon-anticodon pairing rather than the identity of the amino acid being delivered, suggesting flexibility in ribosomal function.

Sid Hecht’s Semi-synthetic tRNA:

  • Development of a method to create tRNAs that incorporate synthetic amino acids, showcasing innovative techniques in molecular biology.

  • Techniques involved RNA ligase and truncated tRNA modifications to allow the attachment of synthetic amino acids.

  • Challenges encountered included skepticism regarding the feasibility of non-natural amino acid research and significant funding limitations in the field.

Modified tRNA Synthetases

  • In-depth exploration of re-engineering tRNA synthetases to enable the incorporation of non-natural amino acids into the protein synthesis pathway.

Peter Schultz's Innovations:

  • Noteworthy contributions in developing techniques for pairing synthetic tRNAs with stop codons, thus enabling specialized incorporation pathways for non-natural amino acids.

  • Emphasis on orthogonality, ensuring that engineered tRNAs and synthetases function without interference from their natural counterparts, thus maintaining cellular integrity.

Orthogonality Explained

  • An essential concept in genetic engineering that demands engineered systems to selectively incorporate only the intended non-natural amino acids at designated sites within the protein sequence.

  • Ensures compatibility: The modified synthetases must be unable to accept natural amino acids or interact with native tRNAs, thereby preventing disruption of normal cellular processes.

Practical Applications of Non-Natural Amino Acids

  • Outline of research stages and experimentation methodologies utilized to optimize the incorporation of synthetic amino acids into E. coli models.

  • Use of selective pressures through positive and negative selection techniques to evolve synthetases with desired specificities for non-natural substrates.

  • Several examples of successfully engineered synthetases that only respond to non-natural amino acids, marking a milestone in synthetic biology.

Examples of Non-Natural Amino Acids Incorporated

  • Constructs used in research that allow researchers to examine various biochemical processes and protein structures.

  • Potential applications range from improving methods in click chemistry, enhancing photocross-linking techniques, and furthering studies in post-translational modifications, thereby enriching the field of protein engineering.

Limitations and Tolerance of the Ribosome

Masahiko Saito’s Experiments:

  • Innovations include a honeycomb diagram designed to represent ribosome tolerance toward non-natural amino acids, setting up a framework for understanding structural implications.

  • Investigations into how specific structural changes to amino acids can affect ribosomal incorporation efficiency, leading to insights into ribosomal behavior.

Virginia Cornish’s Studies:

  • Research focused on the tolerances of ribosomes regarding backbone modifications, assessing how these alterations influence ribosomal activity and protein synthesis fidelity.

Adjusting Ribosomes for Non-Natural Incorporation

Jason Chin’s Approach:

  • Proposal for creating orthogonal ribosomes that preferentially facilitate the incorporation of non-natural amino acids while minimizing interference with release factors crucial for terminating protein synthesis.

  • Modifications to the Shine-Dalgarno sequence to allow targeted manipulation of ribosomes without disrupting the normal functioning of natural ribosomes.

Conclusion

  • Engineering the central dogma offers groundbreaking possibilities for advancements in synthetic biology and the development of novel biotechnological applications.

  • Ongoing research endeavors aim to extend the capabilities of non-natural amino acids through the creation of robust orthogonal systems, fostering innovation in protein design and functional diversity.

  • Discussion included insights into the upcoming quiz, reinforcing key concepts for students before the spring break.