Nucleic Acids and Protein Synthesis PDF

Nucleic acids and Protein Synthesis  Introduction – Each cell of our bodies contains thousands of different proteins. – How do cells know which proteins to synthesize out of the extremely large number of possible amino acid sequences? – the transmission of heredi...

Nucleic acids and Protein Synthesis  Introduction – Each cell of our bodies contains thousands of different proteins. – How do cells know which proteins to synthesize out of the extremely large number of possible amino acid sequences? – the transmission of hereditary information took place in the nucleus, more specifically in structures called chromosomes. – The hereditary information was thought to reside in genes within the chromosomes. – Chemical analysis of nuclei showed chromosomes are made up largely of proteins called histones and nucleic acids.  Nucleic acids Backbones of chromosomes Ribonucleic acids (RNA) Nucleic acids Deoxyribonucleic acids (DNA)  Nucleic acids DNA stores the genetic information of an organism and transmits that information from one generation to another. RNA translates the genetic information contained in DNA into proteins needed for all cellular function. RNA and DNA are unbranched polymers (monomers: nucleotides).  Nucleotide A nucleotide is composed of: Nitrogen-containing bases (amines) Sugars (monosaccharides) Phosphate Phosphate Bases  Sugars (monosaccharide) RNA contains: D-Ribose sugar DNA contains: 2-Deoxy-D-Ribose sugar (without O on carbon 2)  Nucleoside When a N atom of the base forms a glycosidic bond to C1’ (anomeric C) of a sugar. Base + Sugar Nucleoside ß-N-glycosidic bond  Nucleoside To name a nucleoside derived from a pyrimidine base, use the suffix “-idine”. To name a nucleoside derived from a purine base, use the suffix “-osine”. For deoxyribonucleosides, add the prefix “deoxy-”.  Nucleotide A nucleotide forms with the −OH on C5’ of a sugar bonds to phosphoric acid. Phosphate ester bond 5’ 5’ 1’ A nucleotide The name cytidine 5′-monophosphate is abbreviated as CMP.  Nucleotide The name deoxyadenosine 5’-monophosphate is abbreviated as dAMP.  Primary structure of DNA and RNA Polynucleotide Carry all information for protein synthesis. Phosphodiester bond Sequence of nucleotides. Each phosphate is linked to C3’ and C5’ of two sugars.  Primary structure of DNA and RNA A nucleoside = Base + Sugar A nucleotide = Base + Sugar + Phosphate A nucleic acid = A chain of nucleotides Like amino acids (C-terminal and N-terminal): Base sequence is read from the C5’ (free phosphate) end to the C3’ (free hydroxyl) end. -ACGU-  Secondary structure of DNA The DNA model is proposed by 5’ 3’ Watson and Crick in 1953. Two strands of polynucleotide form a double helix structure like a spiral. Sugar phosphate backbone 3D structure Hydrogen bonds link paired bases: Adenine-Thymine (A–T) Guanine-Cytosine (G-C) Sugar-Phosphate backbone is hydrophilic and stays on the outside (bases are hydrophobic). 3’ 5’  Secondary structure of DNA A Purine base always hydrogen bonds with a pyrimidine. Complementary base pairs A-T base pair 2 H bonds G-C base pair 3 H bonds  Higher structure of DNA DNA is coiled around proteins called histones. Histones are rich in the basic amino acids Acidic DNA basic histones attract each other and form a chain of nucleosomes. Core of eight histones Higher structure of DNA Chromatin: Condensed nucleosomes  Higher structure of DNA Chromatin fibers are organized into loops, and the loops into the bands that provide the superstructure of chromosomes.  Chromosome & Gene - DNA molecules contain several million nucleotides, while RNA molecules have only a few thousand. - DNA is contained in the chromosomes of the nucleus, each chromosome having a different type of DNA. - Humans have 46 chromosomes (23 pairs), each made up of many genes. - A gene is the portion of the DNA molecule responsible for the synthesis of a single protein (1000 to 2000 nucleotides).  Difference between DNA & RNA 1. DNA has four bases: A, G, C, and T. RNA has four bases: A, G, C, and U. 2. In DNA: Sugar is 2-deoxy-D-ribose. In RNA: Sugar is D-ribose. 3. DNA is almost always double-stranded (helical structure). RNA is single strand. 4. RNA is much smaller than DNA.  RNA molecules Transmits the genetic information needed to operate the cell. 1. Ribosomal RNA (rRNA) Most abundant RNA – is found in ribosomes: sites for protein synthesis. 2. Messenger RNA (mRNA) Carries genetic information from DNA (in nucleus) to ribosomes (in cytoplasm) for protein synthesis. They are produced in “Transcription” from DNA. 3. Transfer RNA (tRNA) The smallest RNA. Translates the genetic information in mRNA and brings specific Amino acids to the ribosome for protein synthesis.  Functions of DNA 1. It reproduces itself when a cell divides (Replication). 2. It supplied the information to make up RNA, proteins, and enzymes.  Replication Separation of the two original strands and synthesis of two new daughter strands using the original strands as templates. By breaking H-bonds  Replication Replication is bidirectional: takes place at the same speed in both directions. Replication is semiconservative: each daughter molecule has one parental strand and one newly synthesized one. Origin of replication: specific point of DNA where replication begins. Replication fork: specific point of DNA where replication is proceeding. Replication occurs at many places simultaneously along the helix.  Replication Leading strand: is synthesized continuously in the 5’ → 3’ direction toward the replication fork. Lagging strand: is synthesized discontinuously in the 5’ → 3’ direction away from the replication fork. https://www.youtube.com/watch?v=hTF4NZC29mU  Replication Replisomes: assemblies of “enzyme factories”.  Helicases Unwinds the DNA double helix. - Replication of DNA starts with unwinding of the double helix. - Unwinding can occur at either end or in the middle. - Attach themselves to one DNA strand and cause separation of the double helix.  Primases Catalyze the synthesis of primers. Primers: are short nucleotides (4 to 15). - They are required to start the synthesis of both daughter strands. - Primases are placed at about every 50 nucleotides in the lagging strand synthesis.  DNA Polymerase It catalyzes the formation of the new strands. - It joins the nucleoside triphosphates found in the nucleus. - A new phosphodiester bond is formed between the 5’-phosphate of the nucleoside triphosphate and the 3’-OH group of the new DNA strand.  Ligase In formation of lagging strand, small fragments (Okazaki) are join together by ligase enzyme.  Protein Synthesis Gene expression: activation of a gene to produce a specific protein. Only a small fraction (1-2%) of the DNA in a chromosome contains genes. Base sequence of the gene carries the information to produce one protein molecule. Change of sequence New protein  Gene expression Transcription: synthesis of mRNA (messenger RNA) Translation Reverse transcription  Transcription Genetic information is copied from a gene in DNA to make a mRNA. Begins when the section of a DNA that contains the gene to be copied unwinds. Polymerase enzyme identifies a starting point to begin mRNA synthesis. https://www.youtube.com/watch?v=NDIJexTT9j0  Transcription The DNA splits into two strands: Template strand: it is used to synthesize RNA. Coding Strand (Informational strand): it is not used to synthesize RNA. - Transcription proceeds from the 3’ end to the 5’ end of the template. (informational strand, non-template strand) Direction of transcription - When mRNA is released, the double helix of the DNA re-forms.  Transcription C is paired with G, T pairs with A But A pairs with U (not T). Polymerase enzyme moves along the unwound DNA, forming bonds between the bases. RNA Polymerase Section of bases on DNA (template strand): -G–A–A–C–T- Complementary base sequence in -C–U–U–G–A- mRNA:  Transcription Sample Problem 22.6 From the template strand of DNA below, write out the mRNA and informational strand of DNA sequences: Template strand: 3’—C T A G G A T A C—5’ mRNA: 5’—G A U C C U A U G—3’ Informational 5’—G A T C C T A T G—3’ strand:  Translation mRNA (as a carrier molecule) moves out of the nucleus and goes to ribosomes. tRNA converts the information into amino acids. Amino acids are placed in the proper sequence. Proteins are synthesized.  Gene expression Overall function of RAN’s in the cell: facilitate the task of synthesizing protein.  Genetic code Genetic code: language that relates the series of nucletides in mRNA to the amino acids specified. The sequence of nucleotides in the mRNA determines the amino acid order for the protein. Every three bases (triplet) along the mRNA makes up a codon. Each codon specifies a particular amino acid. Codons are present for all 20 amino acids. Genetic code  Genetic code 64 condons are possible from the triplet combination of A, G, C, and U. Codons are written from the 5’ end to the 3’ end of the mRNA molecule UGA, UAA, and UAG, are stop signals. (code for termination of protein synthesis). AUG has two roles: 1. Signals the start of the proteins synthesis (at the beginning of an mRNA). 2. Specifies the amino acid methionine (Met) (in the middle of an mRNA).  tRNA (transfer RNA) tRNA translates the codons into specific amino acids. Serine - It contains 70-90 nucleotides. - The 3’ end, called the acceptor stem and always has the nucleotide ACC and a free OH group that binds a specific amino acid. T loop D loop - Anticodon: a sequence of three nucleotides at the bottom of tRNA, which is complementary to three bases in an mRNA and it can identify the needed amino acid. Anticodon loop A G U Codon on mRNA U C A Transcription Translation  Protein synthesis mRNA attaches to smaller subunit of a ribosome. tRNA molecules bring specific amino acids to the mRNA. Peptide bonds form between an amino acid and the end of the growing peptide chain. The ribosome moves along mRNA until the end of the codon (translocation). The polypeptide chain is released from the ribosome and becomes an active protein. Sometimes several ribosomes (polysome) translate the same strand of mRNA at the same time to produce several peptide chains.  Termination Ribosome encounters a stop condon. No tRNA to complement the termination codon. An enzyme releases the complete polypeptide chain from the ribosome. Amino acids form the three-dimensional structure (active protein).  Translation There are 3 stages in translation: 1. Initiation begins with mRNA binding to the ribosome. 2. Elongation proceeds as the next tRNA molecule delivers the next amino acid, and a peptide bond forms between the two amino acids.  Translation 3. Termination: Translation continues until a stop codon (UAA, UAG, or UGA) is reached and the completed protein is released. Often the first amino acid (methionine) is not needed and it is removed after protein synthesis is complete.  Mutation A heritable change in DNA nucleotide sequence. It changes the sequence of amino acids (structure and function of proteins). Enzyme cannot catalyze. X rays, Overexpose to sun (UV light), Chemicals (mutagens), or Viruses However, some mutations are random events.  Effect of Mutation Somatic cell (nonreproductive cell): Altered DNA will be limited to that cell and its daughter Cancer cells. Germ cell (reproductive cell like an egg or sperm): All new DNA will contain the same default and it is passed on to the next generation. Genetic diseases  Type of Mutations Point (substitution) Mutation The most common Replacement of one base in the coding strand of DNA with another. Different amino acid Frameshift Mutation One or more bases is/are added to or deleted from the normal order of bases in DNA. All the triplets shift over by one base. Different sequence of amino acids  Point Mutation In hemoglobin, substitution of just one amino acid can result in the fatal disease sickle cell anemia. No more amino acids are added. A need protein is not synthesized. The organism may die.  Frameshift Mutation 1. A deletion mutation occurs when one or more nucleotides is/are lost from a DNA molecule. 2. An insertion mutation occurs when one or more nucleotides is/are added to a DNA molecule.  Silent Mutation A silent mutation has a negligible effect to the organism, because the resulting amino acid is identical. The mutation has no effect.  Recombinant DNA Recombinant DNA is synthetic DNA that contains segments from more than one source. Three key elements are needed to form recombinant DNA: 1. A DNA molecule into which a new DNA segment will be inserted. 2. An enzyme that cleaves DNA at specific locations. 3. A gene from a second organism that will be inserted into the original DNA molecule. Recombinant DNA First, bacterial plasmid DNA is cut by the restriction endonuclease EcoRI, which cuts in a specific place. This gives a double strand of linear plasmid DNA with two ends ready to bond, called sticky ends.  Recombinant DNA Then, a second sample of human DNA is cut with the same EcoRI. This forms human DNA segments with sticky ends that are complimentary to the plasmid DNA.  Recombinant DNA Combining the two pieces of DNA (with DNA ligase enzyme) forms DNA containing the new segment. This DNA chain is slightly larger because of its additional segment. This new DNA is re-inserted into a bacterial cell. Large amounts of needed proteins can be synthesized by bacteria. Process of Recombinant DNA Technology Examples of Recombinant DNA Products Examples of Recombinant DNA Products Antisense RNA Strategy Antisense technology exploits oligonucleotide analogs to bind to target RNAs via Watson-Crick by hybridization. Once bound, the antisense agent either disables or induces the degradation of the target RNA. Antisense agents may also be used to alter splicing. Examples of Recombinant DNA Products  Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) amplifies a specific portion of a DNA molecule, producing millions of exact copies. Four elements are needed to amplify DNA by PCR: 1. The segment of DNA that must be copied. 2. Two primers—short polynucleotides that are complementary to the two ends of the segment to be amplified. 3. A DNA polymerase enzyme to catalyze the synthesis of a complementary strand. 4. Nucleoside triphosphates—the source of the A, T, C, and G needed to make the new DNA. HOW TO Use the Polymerase Chain Reaction to Amplify a Sample of DNA Heat the DNA segment to unwind the Step double helix to form single strands. HOW TO Use the Polymerase Chain Reaction to Amplify a Sample of DNA Add primers that are complementary to Step the DNA sequence at either end of the DNA segment. HOW TO Use the Polymerase Chain Reaction to Amplify a Sample of DNA Use a DNA polymerase and added Step nucleotides to lengthen the DNA segment. After each cycle the amount of DNA is doubled, so after 20 cycles, 1,000,000 copies have been made. https://www.youtube.com/watch?v=db0HzFTJtCs Applications of Polymerase Chain Reaction  DNA Fingerprinting The DNA of each individual person is unique, so DNA can be used as a method of identification. Any type of cell (skin, saliva, semen, blood, etc.) can be used to obtain a DNA fingerprint. The DNA is first amplified by PCR, and then cut into fragments by restriction enzymes. The DNA fragments are then separated by size by gel electrophoresis.  DNA Fingerprinting DNA fragments can be visualized on X-ray film after they have been separated: End of presentation