Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation which is critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing immunological defense, storage, transport, cellular communication, movement, and structural support. A protein’s function mostly depends on its ability to recognize and bind other molecules, analogous to a lock and key. Hence the specific activity of each protein depends on its unique three-dimensional architecture. For a protein to be functional, it must fold accurately. Most proteins go through several intermediate forms before folding into the most stable, biologically active structure. Misfolding of proteins has detrimental effects on the overall functioning of the cell. In humans, several diseases are due to the accumulation of misfolded or unfolded proteins. These include cystic fibrosis, Alzheimer’s, Parkinson’s, ALS, and Creutzfeldt-Jakob disease. Proteins are made up of one or more chains of amino acids, called polypeptides. A polypeptide is synthesized as a linear chain which rapid…
Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Mutations that occur at a single nucleotide are called point mutations. When point mutations occur within genes, the consequences can vary in severity depending on what happens to the encoded amino acid sequence. A silent mutation does not change the amino acid identity and will have no effect on an organism. A missense mutation changes a single amino acid, and the effects might be serious if the change alters the function of the protein. A nonsense mutation produces a stop codon that truncates the protein, likely rendering it nonfunctional. Frameshift mutations occur when one or more nucleotides are inserted into or deleted from a protein-coding DNA sequence, affecting all of the codons downstream of the location of the mutation. The most drastic type of mutation, chromosomal alteration, changes the physical structure of a chromosome. Chromosomal alterations can include deletion, duplication, or inversion of large stretches of DNA within a single chromosome, or integration o…
Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use. The central dogma of molecular biology states that DNA contains the information that encodes proteins and RNA uses this information to direct protein synthesis. Different types of RNA are involved in protein synthesis. Based on whether or not they encode proteins, RNA is broadly classified as protein-coding or non-coding RNA. Messenger RNA (mRNA) is the protein-coding RNA. It consists of codons—sequences of three nucleotides that encode a specific amino acid. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are non-coding RNA. tRNA acts as an adaptor molecule that reads the mRNA sequence and places amino acids in the correct order in the growing polypeptide chain. rRNA and other proteins make up the ribosome—the seat of protein synthesis in the cell. During translation, ribosomes move along an mRNA strand where they stabilize the binding of tRNA molecules and catalyze the for…
Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA—unlike genomic DNA—also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene. cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types—transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail—a string of adenine nucleotides—added to its 3’ end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away. Next, reverse transcriptase—a DNA polymerase enzyme from retroviruses—is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3’ end of a chain, a pol…
Silk fibers have been processed and used to create fabrics and threads for centuries. However, the solubilizing of silk fibers, thereby turning it into a versatile pre-polymer solution is a much newer technology. Solubilized silk can be processed in many different ways to create a biocompatible material with controllable mechanical properties.
Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a 'pull-down' assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The …
1Department of Neurobiology, F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, 2Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Genome Campus, 3Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, 4Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research
1Division of Nephrology and Hypertension, and the Center for Kidney Research and Therapeutics at the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, 2Surgery-Organ Transplantation, Northwestern University Feinberg School of Medicine, 3School of Biological Sciences and Medical Engineering, Southeast University
1Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, 2Oncomedic Incorporation, Ciudad de México, 3Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México
1Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, 2Department of Obstetrics and Gynecology, College of Medicine, University of Arkansas for Medical Sciences, 3Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences
1Dipartimento di Biotecnologie, Università degli Studi di Verona, 2Ecology and Evolution, Research School of Biology, The Australian National University, 3Dipartimento di Agraria, SACEG, Università degli Studi di Sassari
1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital
1Department of Anesthesiology, Medical College of Wisconsin, 2Clement J. Zablocki Veterans Affairs Medical Center, 3Department of Surgery, Division of Pediatric Surgery, Children's Research Institute, 4Department of Orthopedic Surgery, Medical College of Wisconsin, 5Deptarment of Anesthesiology, Clement J Zblocki Veteran Affairs Medical Center, 6Department of Medicine, Division of Cardiology, Medical College of Wisconsin
1Protein Crystallization Lab, Emerald Bio, 2Molecular Biology Lab, Emerald Bio, 3Scientific Sales Representative, Emerald Bio, 4Group Leader II, Emerald Bio, 5Group Leader I, Emerald Bio, 6Chair of Advisory Board, Emerald Bio, 7Director of Multi-Target Services, Emerald Bio, 8Senior Project Leader, Emerald Bio, 9Project Leader II & SSGCID Site Manager, Emerald Bio
1Institute for Molecular Biotechnology, RWTH Aachen University, 2Sanofi Deutschland GmbH, 3Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.
1The Smidt Heart Institute, Cedars-Sinai Medical Center, 2Advanced Clinical Biosystems Research Institute, Cedars-Sinai Medical Center, 3Institute of Analytical Chemistry of the Czech Academy of Sciences
1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4The State Key Laboratory of Medical Genetics, Central South University
1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation
1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, 3Swiss Institute of Bioinformatics, 4Clinical Microbiology, University Hospital Basel