Lecture 9#Translation PDF

Notes on Molecular Genetics Lecture 9#Translation - From RNA to protein An mRNA Sequence Is Decoded in Sets of Three Nucleotides Once an mRNA has been produced, by transcription and processing the information present in its nucleotide sequence is used to synthesize a p...

Notes on Molecular Genetics Lecture 9#Translation - From RNA to protein An mRNA Sequence Is Decoded in Sets of Three Nucleotides Once an mRNA has been produced, by transcription and processing the information present in its nucleotide sequence is used to synthesize a protein. Transcription is simple to understand as a means of information transfer: since DNAand RNA are chemically and structurally similar, the DNA can act as a direct template for the synthesis of RNA by complementary base-pairing. The conversion of the information in RNA into protein represents a translation of the information into another language that uses quite different symbols. Moreover, since there are only four different nucleotides in mRNAand twenty different types of amino acids in a protein, this translation cannot be accounted for by a direct one-to-one correspondence between a nucleotide in RNA and an amino acid in protein. The nucleotide sequence of a gene, through the medium of mRNA, is translated into the amino acid sequence of a protein by rules that are known as the genetic code. This code was deciphered in the early 1960s. The sequence of nucleotides in the mRNA molecule is read consecutively in groups of three. RNA is a linear polymer of four different nucleotides, so there are 4 × 4 × 4 = 64 possible combinations of three nucleotides: the triplets AAA, AUA, AUG, and so on. However, only 20 different amino acids are commonly found in proteins. Either some nucleotide triplets are never used, or the code is redundant and some amino acids are specified by more than one triplet. The second possibility is, in fact, the correct one, as shown by the completely deciphered genetic code in Figure 6-50. Each group of three consecutive 1  Notes on Molecular Genetics nucleotides in RNA is called a codon, and each codon specifies either one amino acid or a stop to the translation process. This genetic code is used universally in all present-day organisms. Although a few slight differences in the code have been found, these are chiefly in the DNA of mitochondria. Mitochondria have their own transcription and protein synthesis systems that operate quite independently from those of the rest of the cell, and it is understandable that their small genomes have been able to accommodate minor changes to the code. M-Genetic codon Genetic codon tRNA Molecules Match Amino Acids to Codons in mRNA The translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs), each about 80 nucleotides in length. Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn schematically (Figure 46). The cloverleaf undergoes further folding to form a compact L-shaped structure that is 2  Notes on Molecular Genetics held together by additional hydrogen bonds between different regions of the molecule (Figure 50). Figure 50: tRNA molecule In this series of diagrams, the same tRNA molecule—in this case a tRNA specific for the amino acid phenylalanine (Phe)—is depicted in various ways. (A) The cloverleaf structure, a convention used to show the complementary base-pairing (red lines) that creates the double-helical regions of the molecule. The anticodon is the sequence of three nucleotides that base-pairs with a codon in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3′ end of the tRNA. tRNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been synthesized. For example, the bases denoted Ψ (for pseudouridine—see Figure 6-43) and D (for dihydrouridine—see Figure 6-55) are derived from uracil. (B and C) Views of the actual L-shaped molecule, based on x-ray diffraction analysis. Although a particular tRNA, that for the amino acid phenylalanine, is depicted, all other tRNAs have very similar structures. (D) The linear nucleotide sequence of the molecule, color-coded to match A, B, and C. We have seen in the previous section that the genetic code is redundant; that is, several different codons can specify a single amino acid (Figure 50). This redundancy implies either that there is more than one tRNA for many of the amino acids or that some tRNA molecules can base-pair with more than one codon. 3  Notes on Molecular Genetics Figure 50: The genetic code. The standard one-letter abbreviation for each amino acid is presented below its three-letter abbreviation (see Panel 3-1, pp. 132–133, for the full name of each amino acid and its structure). By convention, codons are always written with the 5′-terminal nucleotide to the left. Note that most amino acids are represented by more than one codon, and that there are some regularities in the set of codons that specifies each amino acid. Codons for the same amino acid tend to contain the same nucleotides at the first and second positions, and vary at the third position. Three codons do not specify any amino acid but act as termination sites (stop codons), signaling the end of the protein-coding sequence. One codon—AUG—acts both as an initiation codon, signaling the start of a protein-coding message, and also as the codon that specifies methionine. Two regions of unpaired nucleotides situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein synthesis. One of these regions forms the anticodon, a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule. The other is a short single-stranded region at the 3′ end of the molecule; this is the site where the amino acid that matches the codon is attached to the tRNA. The RNA Message Is Decoded on Ribosomes Ribosomes operate with remarkable efficiency: in one second, a single ribosome of a eucaryotic cell adds about 2 amino acids to a polypeptide chain; the ribosomes of bacterial cells operate even faster, at a rate of about 20 amino acids per second. A ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A-site, the P-site, and the E-site) are for tRNAs (Figure 55). 4  Notes on Molecular Genetics A tRNA molecule is held tightly at the A- and P-sites only if its anticodon forms base pairs with a complementary codon (allowing for wobble) on the mRNA molecule that is bound to the ribosome. Figure 55 The RNA-binding sites in the ribosome. Each ribosome has three binding sites for tRNA: the A-, P-, and E-sites (short for aminoacyl-tRNA, peptidyl-tRNA, and exit, respectively) and one binding site for mRNA. (A) Structure of a bacterial ribosome with the small subunit in the front (dark green) and the large subunit in the back (light green). Both the rRNAs and the ribosomal proteins are shown. tRNAs are shown bound in the E-site (red), the P-site (orange) and the A-site (yellow).Although all three tRNA sites are shown occupied here, during the process of protein synthesis not more than two of these sites are thought to contain tRNA molecules at any one time (see Figure 6-65). (D) Highly schematic representation of a ribosome. 5  Notes on Molecular Genetics 6  Notes on Molecular Genetics Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis The translation of an mRNA begins with the codon AUG, and a special tRNA is required to initiate translation. This initiator tRNA always carries the amino acid methionine (in bacteria, a modified form of methionine—formylmethionine—is used) so that all newly made proteins have methionine as the first amino acid at their N-terminal end, the end of a protein that is synthesized first. This methionine is usually removed later by a specific protease. The initiator tRNA has a nucleotide sequence distinct from that of the tRNA that normally carries methionine. The mechanism for selecting a start codon in bacteria is different. Bacterial mRNAs have no 5′ caps to tell the ribosome where to begin searching for the start of translation. Instead, each bacterial mRNA contains a specific ribosome-binding site (called the Shine-Dalgarno sequence, named after its discoverers) that is located a few nucleotides upstream of the AUG at which translation is to begin. This nucleotide sequence, with the consensus 5′-AGGAGGU-3′, forms base pairs with the 16S rRNA of the small ribosomal subunit to position the initiating AUG codon in the ribosome. A set of translation initiation factors orchestrates this interaction, as well as the subsequent assembly of the large ribosomal subunit to complete the ribosome. Unlike a eucaryotic ribosome, a bacterial ribosome can therefore readily assemble directly on a start codon that lies in the interior of an mRNA molecule, so long as a ribosome-binding site precedes it by several nucleotides. As a result, bacterial mRNAs are often polycistronic—that is, they encode several different proteins, each of which is translated from the same mRNA molecule (Figure 57). In contrast, a eucaryotic mRNA generally encodes only a single protein. 7  Notes on Molecular Genetics Figure 57 Structure of a typical bacterial mRNA molecule. Unlike eucaryotic ribosomes, which typically require a capped 5′ end, procaryotic ribosomes initiate transcription at ribosome-binding sites (Shine-Dalgarno sequences), which can be located anywhere along an mRNA molecule. This property of ribosomes permits bacteria to synthesize more than one type of protein from a single mRNA molecule. Stop Codons Mark the End of Translation The end of the protein-coding message is signaled by the presence of one of three codons (UAA, UAG, or UGA) called stop codons. These are not recognized by a tRNA and do not specify an amino acid, but instead signal to the ribosome to stop translation. Proteins known as release factors bind to any ribosome with a stop codon positioned in the A site, and this binding forces the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA. - Gene Expression Can Be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein SELF STUDY ITEM If differences among the various cell types of an organism depend on the particular genes that the cells express, at what level is the control of gene expression exercised? As we saw in the last chapter, there are many steps in the pathway leading from DNA to protein, and all of them can in principle be regulated. Thus a cell can control the proteins it makes by (1) controlling when and how often a given gene is transcribed (transcriptional control), (2) controlling how the RNA transcript is spliced or otherwise processed (RNA 8  Notes on Molecular Genetics processing control), (3) selecting which completed mRNAs in the cell nucleus are exported to the cytosol and determining where in the cytosol they are localized (RNA transport and localization control), (4) selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control), (5) selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control), or (6) selectively activating, inactivating, degrading, or compartmentalizing specific protein molecules after they have been made (protein activity control) (Figure 58). Figure 58 steps at which eucaryotic gene expression can be controlled. Controls that operate at steps 1 through 5 are discussed in this chapter. Step 6, the regulation of protein activity, includes reversible activation or inactivation by protein phosphorylation (discussed in Chapter 3) as well as irreversible inactivation by proteolytic degradation. For most genes transcriptional controls are paramount. This makes sense because, of all the possible control points illustrated in Figure 7-5, only transcriptional control ensures that the cell will not synthesize superfluous intermediates. In the following sections we discuss the DNA and proteincomponents that perform this function by regulating the 9  Notes on Molecular Genetics initiation of genetranscription. We shall return at the end of the chapter to the additional ways of regulating gene expression. - OMICS - SELF STUDY ITEM The suffex OMIC refers to the study of the complete set of molecules within a biological system, including genomics, transcriptomics, proteomics, and metabolomics to identify genetic factors and metabolic pathways that play important roles in the growth and development of biological systems that would be affected by environmental conditions, diseases ------etc. By integrating multiple OMIC datasets, researchers can gain a more comprehensive understanding of the molecular mechanisms underlying living cells growth and development as well as responses to environmental stresses. OMIC approaches involve the comprehensive analysis of various biological molecules, including DNA, RNA, proteins, and metabolites, to gain insights into the molecular mechanisms underlying cell growth and development. These approaches have revolutionized the field of cell biology by enabling the simultaneous analysis of multiple molecules and providing a systems-level understanding of cell processes. - Genomics involves the study of the entire genome of an organism, including the identification of genes and their functions. The availability of genome sequences for several organisms enabled the identification of key genes and pathways associated with important traits. Studying the genome involves a range of techniques that allow researchers to analyze the DNA content of an organism. Some common techniques used in genome studies include: 10  Notes on Molecular Genetics 1. DNA Sequencing: o Sanger Sequencing: The traditional method for sequencing DNA. o Next-Generation Sequencing (NGS): High-throughput sequencing technologies that enable rapid sequencing of large amounts of DNA. 2. Genome Assembly: o De Novo Assembly: Building a genome sequence from scratch without a reference genome. o Reference-Based Assembly: Using a known reference genome to align and assemble sequenced reads. 3. Genome Annotation: o Identification of Genes and Functional Elements: Tools like GeneMark, AUGUSTUS, and BLAST are used to predict genes and annotate functional elements. 4. Comparative Genomics: o Whole-Genome Alignment: Comparing genomes to identify similarities and differences. o Synteny Analysis: Studying conserved genomic arrangements across species. 5. Metagenomics: o Analysis of Microbial Communities: Studying the genomes of microbial communities in environmental samples. 6. Functional Genomics: o Gene Knockout/Knockdown: Disrupting gene function to study its effects. 7. Phylogenomics: 11  Notes on Molecular Genetics o Phylogenetic Analysis: Studying evolutionary relationships using genomic data. These techniques, along with advancements in bioinformatics and computational biology, help researchers gain insights into the functions of genes, regulatory elements, and pathways within the genome. - Transcriptomics involves the analysis of the entire transcriptome of an organism, including the identification of all the expressed genes and their levels of expression. Transcriptomics has been used to identify genes and pathways associated with particular diseases and/or environmental conditions. Transcriptomics focuses on studying RNA expression patterns to understand gene expression levels, alternative splicing, and regulatory mechanisms within cells or tissues. Various techniques are employed in transcriptomic analysis, some of which include: 1. RNA Sequencing (RNA-Seq): o Quantification of Gene Expression: Measures the abundance of RNA transcripts in a sample. o Identification of Alternative Splicing Events: Detects different isoforms of genes. 2. Microarray Analysis: o Gene Expression Profiling: Measures the expression levels of a large number of genes simultaneously. o Comparative Expression Studies: Compares gene expression across different conditions or tissues. 3. Quantitative Real-Time PCR (qRT-PCR): o Validation of Gene Expression: Confirms gene expression patterns observed in RNA-Seq or microarray studies. These techniques help researchers gain insights into the dynamic landscape of gene expression and regulation, providing valuable information about cellular processes, developmental pathways, and disease mechanisms. 12  Notes on Molecular Genetics - Proteomics involves the study of the entire proteome of an organism, including the identification of all the proteins and their functions. Proteomics has been used to identify proteins associated with stress response. Proteomics is the study of the entire set of proteins expressed by a cell, tissue, or organism. Proteomic analysis involves a variety of techniques to study protein structure, function, and interactions. Some common techniques used in proteomic analysis include: 1. Mass Spectrometry (MS): o Liquid Chromatography-Mass Spectrometry (LC-MS): Separates and identifies proteins based on mass-to-charge ratios. o Tandem Mass Spectrometry (MS/MS): Fragmentation of peptides for sequencing and identification. 2. Two-Dimensional Gel Electrophoresis (2D-PAGE): o Separation of Proteins: Separates proteins based on isoelectric point and molecular weight for analysis. 3. Protein Identification: o Database Search: Matches experimental mass spectra to known protein sequences for identification. o De Novo Sequencing: Determines protein sequences directly from mass spectra without a reference database. 4. Protein Structure Analysis: 13  Notes on Molecular Genetics o X-Ray Crystallography and NMR Spectroscopy: Techniques used to determine protein structures at atomic resolution. 5. Bioinformatics Analysis: o Database Search Tools: Analyze and interpret proteomic data for functional insights and pathway analysis. These techniques play a crucial role in understanding protein expression, structure, function, interactions, and post-translational modifications, providing valuable insights into cellular processes, diseases, and biological systems. - Metabolomics involves the study of the entire metabolome of an organism, including the identification of all the metabolites and their functions. Metabolomics has been used to identify metabolites associated with particular diseases and/or environmental conditions. Metabolomics is the study of small molecules, known as metabolites, within cells, tissues, or organisms. It aims to understand the metabolic processes and pathways in biological systems. Various techniques are used in metabolomic analysis to identify and quantify metabolites. Some common techniques include: 1. Mass Spectrometry (MS): o Gas Chromatography-Mass Spectrometry (GC-MS): Analyzes volatile and thermally stable compounds. o Liquid Chromatography-Mass Spectrometry (LC-MS): Separates and identifies metabolites based on mass-to-charge ratios. o Tandem Mass Spectrometry (MS/MS): Fragmentation of metabolites for structural elucidation. 2. Nuclear Magnetic Resonance (NMR) Spectroscopy: 14  Notes on Molecular Genetics o High-Resolution Analysis: Provides information on the chemical structure and concentration of metabolites. 3. Targeted Metabolomics: o Quantification of Specific Metabolites: Focuses on measuring a predefined set of metabolites with known biological relevance. 4. Isotope Tracing: o Stable Isotope Labeling: Tracks the fate of labeled metabolites through metabolic pathways to study fluxes and metabolic transformations. These techniques help researchers unravel the metabolic profiles of biological systems, providing insights into physiological conditions, disease mechanisms, and responses to external stimuli such as drugs or environmental factors. - The term epigenome is derived from the Greek word epi which literally means "above" the genome. The epigenome consists of chemical compounds that modify, or mark, the genome in a way that tells it what to do, where to do it, and when to do it. Different cells have different epigenetic marks. These epigenetic marks, which are not part of the DNA itself, can be passed on from cell to cell as cells divide, and from one generation to the next. The epigenome is the collection of all of the epigenetic marks on the DNA in a single cell. The epigenomic marks different between different cell types. So, a blood cell will have different marks or modifications than a liver cell. The epigenomic modifications, the whole collection of all of the epigenetic marks on 15  Notes on Molecular Genetics my blood cell DNA should be more similar to all of the marks on your blood cell DNA than to the collection of all the marks on my liver cell DNA. So this is a way of defining a particular type of cell. Now due to individual differences my epigenome will differ from your epigenome even in the same tissue. It's those differences that make us all individuals, and we'll see even greater changes in a state of disease. So a comparison of a normal cell and all of its epigenetic marks, or the epigenome of that cell, will differ from the diseased state of that same cell type. And we can use these differences to figure out mechanisms of disease. 16