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Proteins and Proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
One strand of DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementary with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3′ → 5′, the coding (non-template) strand and newly formed RNA can also be used as reference points, so transcription can be described as occurring 5′ → 3′. This produces an RNA molecule from 5′ → 3′, an exact copy of the coding strand except that thymines are replaced with uracils and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose in its sugar-phosphate backbone. Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of specific mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene. Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond to short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or because of the chromatin structure.
Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
One strand of DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementary with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3′ → 5′, the coding (non-template) strand and newly formed RNA can also be used as reference points, so transcription can be described as occurring 5′ → 3′. This produces an RNA molecule from 5′ → 3′, an exact copy of the coding strand except that thymines are replaced with uracils and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose in its sugar-phosphate backbone. Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene. Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure.
Biomolecular Processing and Molecular Electronics
Published in Sergey Edward Lyshevski, Molecular Electronics, Circuits, and Processing Platforms, 2018
The transcription and translation processes are illustrated in Figure 3.18. Transcription results in nucleotide-to-nucleotide transfer of coded information from DNA to RNA. RNA synthesis on a DNA template is catalyzed by RNA polymerase. Promoters (specific nucleotides sequences flanking the start of a gene) signal the initiation of mRNA synthesis. Transcription factors (proteins) help RNA polymerase recognize promoter sequences and bind to the RNA. Transcription continues until the RNA polymerase reaches the termination (stop) sequence of nucleotides on the DNA template. As the mRNA peels away, the DNA double helix re-forms. Translation results in the code transfer from RNA nucleotides to polypeptide amino acids (transfer RNA interprets the genetic code during translation, and each kind of tRNA brings a specific amino acid to the ribosome). Transfer RNA molecules pick up specific amino acids and line up by means of their anticodon triplets at complementary codon sites on the mRNA molecule. The ATP process is catalyzed by aminoacryl-tRNA synthetase enzymes. The ribosome controls the coupling of tRNA to mRNA codons. They provide a site for the binding of mRNA, as well as P and A sites (peptidyl-tRNA and aminoacyl-tRNA sites) for holding adjacent tRNA as amino acids are linked in the growing polypeptide chain. There are three major stages—initiation (integrates mRNA with tRNA with the attached first amino acid), elongation (polypeptide chain is completed adding amino acids attached to its tRNA by binding and translocation tRNA and mRNA along the ribosome), and termination (termination codonds cause the protein release freeing the polypeptide chain and dislocation of the ribosome subunits). Several ribosomes can read a single mRNA, forming polyribosome clusters. Complex proteins usually undertake one or several changes during and after translation that affect their 3D structures. This leads to the cell transitional dynamics.
Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Published in Preparative Biochemistry & Biotechnology, 2023
Kashif Maseh, Syed Farhat Ali, Shazeel Ahmad, Naeem Rashid
Heterologous gene expression in E. coli is a common method to produce recombinant proteins as it is a well-studied expression host with short doubling time and ability to express recombinant proteins at high rate.[19] In this study, we used E. coli BL21 CodonPlus (DE3) RIL strain for expression of Pca-pol gene. This strain contains rare-codon transfer RNAs. BL21 strain has also been reported to have better performance in high cell density cultures.[20]Pca-pol gene was cloned in plasmid pET28a. pET expression system is based on T7 promoter and requires T7 RNA polymerase from DE3 strain.[21] The resulting recombinant Pca-Pol contained N-terminal His-tag. Affinity tags such as His-tag facilitate the process of protein purification by affinity chromatography.[22] This can lead to greater recovery of the required recombinant protein, as in our case, IMAC purification recovered 92% of the total activity in the purified fraction.
Differentially expressed long-chain noncoding RNAs in human neuroblastoma cell line (SH-SY5Y): Alzheimer’s disease cell model
Published in Journal of Toxicology and Environmental Health, Part A, 2019
Ming Zhang, Yuan-Qing Zhang, Xie-Ze Wei, Charles Lee, Dong-Sheng Huo, He Wang, Zhi-Ying Zhao
To identify potential lncRNAs and the moieties that these constituents regulate, GO analysis was performed for the differentially expressed lncRNAs to determine which are enriched in GO terms of biological process, cellular component and molecular function as illustrated in Figure 5. The major GO enrichment terms focused on the biological process was categorized as follows: 1) regulation of transcription; 2) DNA-templated; 3) transcription; 4) signal transduction; 5) transport; 6) phosphorylation; 7) positive regulation of transcription from RNA polymerase II promoter; 8) cell cycle; 9) oxidation-reduction process; 10) and nervous system development. The major GO enrichment terms in the present study in the cellular component category were as follows: 1) membrane; 2) nucleus; 3) cytoplasm; 4) cytosol; 5) integral component of membrane; 6) extracellular exosome; 7) mitochondrion; 8) extracellular region; 9) cytoskeleton; 10) endoplasmic reticulum. The major GO enrichment terms in the molecular function category were as follows: 1) protein binding; 2) metal ion binding; 3) nucleotide binding; 4) DNA binding; 5) ATP binding; 6) transferase activity; 7) nucleic acid binding; 8) RNA binding; 9) zinc ion binding; and 10) hydrolase activity.
Effects of cross-fostering and developmental exposure to mixtures of environmental contaminants on hepatic gene expression in prepubertal 21 days old and adult male Sprague-Dawley rats
Published in Journal of Toxicology and Environmental Health, Part A, 2019
D. Desaulniers, N. Khan, C. Cummings-Lorbetskie, K. Leingartner, G-H. Xiao, A. Williams, C.L. Yauk
Total RNA (200 ng) from exposed and control groups, as well as universal reference total RNA (Agilent Technologies) were employed to synthesize complementary DNA (cDNA) and cyanine labeled complementary RNA (cRNA), using Low Input Quick Amp Labeling Kit (Agilent Technologies) according to manufacturer’s instruction. Cyanine-labeled cRNAs were synthesized using T7 RNA polymerase and in vitro transcription kits (Agilent Technologies). cRNA was then purified using RNeasy Mini Kits (Qiagen). Cyanine 5-CTP (Cy-5) was used to label the sample, and universal reference RNAs were labeled with cyanine 3-CTP (Cy-3).