Transcription & Translation PDF

POLYNUCLEOTID ES 1  OVERVIEW Nucleic acids(DNA & RNA) contain: Carbon Hydrogen Oxygen Nitrogen Phosphorus Conserved through evolution in all organisms, storing hereditary information. DNA contains instructions for synthesis of proteins, deciding seq...

POLYNUCLEOTID ES 1  OVERVIEW Nucleic acids(DNA & RNA) contain: Carbon Hydrogen Oxygen Nitrogen Phosphorus Conserved through evolution in all organisms, storing hereditary information. DNA contains instructions for synthesis of proteins, deciding sequences of amino acids in polypeptides through process known as transcription & translation Nucleic acids are made up of nucleotides Consisting of a pentose sugar (deoxyribose or ribose) Nitrogenous base (adenine, cytosine, guanine, and thymine or uracil) Phosphate group Linked together through Phosphodiester Bonds 2  DNA VS RNA DNA carries genetic blueprint passed from parent to offspring via cell division DNA has double helix structure with 2 strands running in opposite directions, connected by H-bonds and complementary to each other. (A-T) and (C-G) RNA is single stranded: messenger (mRNA) ribosomal (rRNA) transfer (tRNA) Central Dogma of Life: Genes contain coded DNA instructions Instructions tell cells how to build proteins Proteins help determine organism’s characteristics 3 DNA VS RNA DNA remains safe in nucleus serving as a template to make multiple RNA copies RNA travels to ribosomes with instructions for how to assemble proteins in cytoplasm 4  RNA TYPES Messenger RNA (mRNA): molecule that carries copy of instructions from nucleus to ribosomes in cytoplasm Ribosomal RNA (rRNA): makes up subunits of ribosomes, which is site of protein synthesis Transfer RNA (tRNA): carries amino acids to ribosome and matches them to coded mRNA message during assembly of a protein 1 DNA molecule contains thousands of genes Only those expressed are copied into RNA Differentiation 5  CENTRAL DOGMA OF LIFE 6  OVERVIEW Gene: Molecular unit of heredity capable of replication, expression and mutation Ethical, legal and health implications given technological advances in field of Genetics. 4 nucleotides can generate sequences of DNA that specify polymers of amino acids Sequences can be transcribed into mRNA and translated into proteins, which perform a variety of functions for the cell Genetic code: includes DNA alphabet (A, T, C, G), RNA alphabet (A, U, C, G) and polypeptide alphabet (20 amino acids). Central Dogma: flow of genetic information from DNA to mRNA to protein. Transcription: genes are used to make mRNA. Translation: mRNA is used to direct synthesis of proteins. Also involves tRNA & rRNA 7  OVERVIEW 2 Genetic code involves an RNA codon consisting of 3 consecutive nucleotides that specify 1 amino acid or the release of newly formed polypeptide chain. Code is degenerate, some amino acids specified by more than 1 codon (synonyms). The genetic code is universal to almost all organisms on Earth (Except: mitochondria & some microbes). Deviations from scheme of Central Dogma include retrovirus (HIV) which stores genetic information in single-strand RNA (not DNA). RNA used as template by enzyme Reverse Transcriptase to synthesize DNA once infect host cell. Some RNA genome viruses never go through DNA step, they use RNA polymerase that is virally encoded. 8  INTRO messengerRNA: mobile molecular copy of 1 or more genes. Translation of mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 amino acids. Each amino acid is defined by a 3 nucleotide sequence called the triplet codon. Different amino acids possess different chemical and structural properties. Variations in amino acid sequences results in variety of protein structure and function. 9  CENTRAL DOGMA OF LIFE Central Dogma: flow of genetic information in cells goes from genes of DNA to mRNA that specifies sequence of amino acids into proteins. Transcription of DNA to RNA involves one nucleotide being added to mRNA strand for every nucleotide read in DNA strand. Translation involves 3 mRNA nucleotides corresponding to 1 amino acid in polypeptide sequence. Colinear: nucleotides 1-3 are amino acid #1, nucleotides 4-6 are amino acid #2, etc… 1 0 GENETIC CODE 11  UNIVERSAL CODONS There are 64 possible nucleotide triplets (4^3), which is more than 20 amino acids. Genetic code is degenerate, an amino acid can be encoded by more than 1 nucleotide triplet (codon). Crick & Brenner used a chemical mutagen to insert 1, 2 or 3 nucleotides into gene of virus. Only synthesized a functional protein when 3 nucleotides were inserted. An insertion or deletion of 1 or 2 nucleotides to the reading frame, alters the message for every subsequent amino acid resulting in… When 3 nucleotides are inserted at once an extra amino acid is generated during translation, but the integrity of the rest of the protein is maintained. 1 2  GENETIC CODE Nonsense codons: also known as stop codons, are able to terminate protein synthesis, releasing the polypeptide from the translation machinery. Start codon: AUG specifies the amino acid methionine, but it also serves to initiate translation. Reading frame is set by its inclusion near the 5’ end of mRNA. The universality of the genetic code is strong evidence that all life on Earth shares a common origin. Could transfer sequence of mRNA from 1 organism to another and have same product made even if normally not synthesized by organism that received inserted mRNA. 1 3 MUTATIONS 14  POINT MUTATIONS Substitution: type of mutation where one base is replaced by another Transition: purine replaced by a purine or pyrimidine replaced by a pyrimidine T is supposed to pair with A, but instead A is replaced by G Transversion: purine replaced by pyrimidine or vice versa. T is supposed to pair with A, but instead A is replaced by C Point mutation: only one base pair is affected Silent: change in sequence of nucleotide does not change amino acid Codon CCC is substituted by CCA. They both make Proline This occurs because the genetic code is degenerate Missense: Substitution which changes the resulting amino acid Codon CAC (His) is substituted by CAA (Gln) Very significant if occurs to Start codon (AUG) Nonsense: change in base that results in making of the stop codon. Codon UAC (Tyr) is substituted by UAA (Stop) This causes the ribosome to end the mRNA’s translation earlier = shorter protein 15  FRAMESHIFT MUTATIONS Reading Frame: divides the sequence of nucleotides in consecutive triplets. Frameshift mutations: moves the nitrogenous bases to accommodate for an insertion or deletion of bases. Most of the time results in a change of codons. Insertion: the addition of 1+ base shifts the reading frame from the left to the right. Deletion: the removal of 1+ base shifts the reading frame from the right to the left. If the bases removed or inserted are in multiples of 3, and at the beginning of the codon, it does not result in a significant change of the resulting protein If the insertion or deletion occurs at the end, it will also not significantly affect the protein. 16 TRANSCRIPTIO N 17  TRANSCRIPTION Transcription: process of copying base sequence from DNA to complementary RNA Enzyme RNA polymerase reads nucleotides from DNA template in a 3’ to 5’ direction It assembles the complementary transcript in a 5’ to 3’ direction. Since it is synthesizing an RNA strand, it is pairing Uracil (U) with Adenine (A), instead of Thymine (T). mRNA synthesis is initiated when RNA Polymerase binds at a promoter sequence on DNA template Transcription continues (elongation) until RNA Pol reaches a stop or terminator sequence at end of gene Termination frees mRNA by the formation of a hairpin 18  CONCURRENT After termination, the process of transcription is complete. By the time termination occurs, prokaryotic transcript would have already begun synthesis of numerous copies of encoded protein because processes occur at same time. Transcription, translation and mRNA degradation are possible because they all occur in the same 5’ to 3’ direction and because there is no membrane compartment separating the processes in the nucleoid. Multiple polymerases can transcribe a single bacterial gene Multiple ribosomes can translate mRNA transcripts into polypeptides As a result, a specific protein can rapidly reach high concentrations in bacterial cell 19  OVERVIEW Before pre-mRNA is translated into protein, it is modified by undergoing splicing Splicing: introns sequences are removed & exons, which encode proteins and will be expressed are reconnected to form final mRNA Some pre-mRNA molecules are cut and spliced in different ways in different tissues. 1 Gene can produce many different mRNA molecules 20 TRANSLATION 21  OVERVIEW After info in gene has gone from pre-mRNA to mRNA it is ready to be translated into a polypeptide Ribosomes consist of small and large subunits of protein and rRNA, which bind mRNA. Many ribosomes can work on same mRNA at a time. Translation begins at initiating AUG on mRNA, specifying 1st amino acid in polypeptide: methionine Each amino acid is carried to ribosome by attaching to specific molecule of tRNA tRNA has amino acid attachment site at 1 end & anticodon sequence that interacts with mRNA codon through complementary base pairing at other end. Amino-acid charging enzymes ensure correct amino acid attached to correct tRNA. Sequential amino acids are linked by peptide bonds. mRNA is translated, undergoing elongation until stop/nonsense codon is reached. A release factor dissociates components and frees new polypeptide. Folding of protein occurs during and after translation. Once polypeptide is synthesized, its role as a protein is established. 22  PROTEIN SYNTHESIS MACHINERY Synthesis of proteins consumes more energy than any other metabolic process After water, proteins make up most of the mass in a cell as they perform every function for it. Translation involves decoding mRNA into amino acids (50-1000) that are covalently bonded, peptide bonds to create proteins. Each amino acid has an amino (NH2) + carboxyl (COOH) group. This reaction is catalyzed by ribosomes and generates 1 H2O (Hydrolysis or Dehydration) 23  TRANSLATION Ribosome attaches to an mRNA molecule in cytoplasm As each codon pass through ribosome tRNA brings amino acid Ribosome attaches amino acids to growing chain Each tRNA carries just 1 kind of amino acid Each tRNA has 3 unpaired bases called anticodon Anticodon is complementary to a codon on mRNA Ribosome has 2nd binding site for tRNA of next codon Ribosomes form covalent bond called peptide bonds between amino acids to create a polypeptide. Chain continues to grow until reach stop codon releasing mRNA and polypeptide 24  TRANSLATION R 25  3 TYPES OF RNA INVOLVED IN TRANSLATION mRNA carries coded message that directs process from nucleus to ribosomes tRNA molecules deliver amino acids to ribosomes to carry out translation Ribosomes are made up of proteins and rRNA molecules which carry out reactions of joining amino acids together. Overall RNA carries the genetic code & translates it too Gene codes for traits that make up a characteristic Proteins can be enzymes which catalyze a chemical reaction Catalyze: start or speed up chemical reaction by lowering the Ea Overall proteins build or operate components of living cells. 26