Selective Topics in Biochemistry 4th Stage PDF

Selective Topics in Biochemistry 4th stage Prepared by Dr. Amany M. Hamed Biochemistry Laboratory, Faculty of Science, Sohag University  Biochemistry of Nucleic Acids 4th stage Prepared b...

Selective Topics in Biochemistry 4th stage Prepared by Dr. Amany M. Hamed Biochemistry Laboratory, Faculty of Science, Sohag University  Biochemistry of Nucleic Acids 4th stage Prepared by Dr. Amany M. Hamed Biochemistry Laboratory, Faculty of Science, Sohag University  Nucleic acids are required for the storage and expression of genetic information.  There are two chemically distinct types of Nucleic acids: Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).  DNA: the storehouse of genetic information, is present not only in the nucleus of eukaryotic organisms, but also in mitochondria and the chloroplasts of the plants. The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication.  Nucleotides: are the monomeric units of the nucleic acids, DNA and RNA.  Each nucleotide is consist of a heterocyclic nitrogenous base, a sugar, and one, two or three phosphate.  DNA contains the purine bases adenine (A) and guanine (G). Also the pyrimidine bases cytosine (C) and thymine (T), the sugar is deoxyribose.  RNA contains A, G, and C, but it has uracil (U) instead of T, the sugar is ribose.  Bases present in DNA and RNA Both DNA and RNA contain the same purine bases: Adenine (A) and Guanine (G). Both DNA and RNA contain the pyrimidine cytosine (C), but they differ in their second pyrimidine base : DNA contains thymine (T) , while RNA contains uracil (U). The atoms in the rings of the bases are numbered 1 to 6 in pyrimidines and 1 to 9 in purines. Numbering of bases is unprimed Sugars present in DNA and RNA o Pentoses (5-C sugars) o Numbering of sugars is primed  Nucleosides Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic bond purines are bonded to C1 of the sugar at their N9 atoms. pyrimidines are bonded to C1 of the sugar at their N1 atoms. If the sugar is D-ribose, a ribonucleoside is produced , if the sugar is 2-deoxyribose a deoxyribonucleoside is produced. In DNA: Deoxyadenosine, Deoxyguanosine, Deoxycytidine, and Deoxthymidine In RNA: Adenosine, Guanosine, Cytidine, and Uridine Deoxyadenosine  Phosphate Group o Mono, di- or triphosphates o Phosphates can be bonded to either C3 or C5 atoms of the sugar  Nucleotides  A nucleotide is a nucleoside with an inorganic phosphate attached to a 5-hydroxyl group of the sugar in ester linkage.  The nitrogenous base is linked by an N-glycosidic bond to the anomeric carbon of the sugar, either ribose or deoxyribose  The names and abbreviations of nucleotides specify the base, the sugar, and the number of phosphates attached.  Nucleotide In deoxynucleotides, the prefix “d” precedes the abbreviation. For example, ADP is Adenosine diphosphate (the base Adenine attached to a ribose that has two phosphate groups) and dATP is deoxyadenosine triphosphate (the base adenine attached to a deoxyribose with three phosphate groups). DNA is a polymer of deoxyribonucleotide RNA is a polymer of ribonucleotide - Deoxyadenosine triphosphate (dATP) Adenosine triphosphate (ATP) - Deoxyguanosine triphosphate (dGTP) Guanosine triphosphate (GTP) - Deoxycytidine triphosphate (dCTP) Cytidine triphosphate (CTP) - Deoxythymidine triphosphate (dTTP) Uridine triphosphate (UTP)  Nucleotide o Nucleotide triphosphates are the building blocks of DNA and RNA. o Polynucleotides such as DNA and RNA are linear sequences of nucleotides linked by 3- to 5- phosphodiester bonds between the sugars. o All cells require energy to ensure their survival and reproduction. The cells get the energy by converting nutrients into a chemically useful form of energy. The most important form of chemical energy is adenosine triphosphate (ATP). o Important components of coenzymes FAD, NAD and Coenzyme A Bases in DNA make hydrogen bonds PURINE PYRIMIDINE PURINE PYRIMIDINE  DNA Structure  DNA is a poly deoxyribonucleotide that contain many monodeoxyribonucleotide covalently linked by phosphodiester bonds.  Phosphodiester bonds join the 3´-OH group of one nucleotide to the 5´-OH group of the next nucleotide.  o With the exception of a few viruses that contain single-stranded DNA, DNA exist as a double- stranded molecules in which the two strands wind around each other forming a double helix. A segment of one strand of a DNA molecule o As proposed by Watson and Crick, each DNA molecule consists of two polynucleotide chains joined by hydrogen bonds between the bases. In each base pair, a purine on one strand forms hydrogen bonds with a pyrimidine on the other strand. In one type of base pair, adenine on one strand pairs with thymine on the other strand. o This base pair is stabilized by two hydrogen bonds. The other base pair, formed between guanine and cytosine, is stabilized by three hydrogen bonds. As a consequence of base-pairing, the two strands of DNA are complementary, that is, adenine on one strand corresponds to thymine on the other strand, and guanine corresponds to cytosine.  The concept of base-pairing proved to be essential for determining the mechanism of DNA replication (in which the copies of DNA are produced that are distributed to daughter cells) and the mechanisms of transcription and translation (in which mRNA is produced from genes and used to direct the process of protein synthesis).  Clearly, as Watson and Crick suggested, base-pairing allows one strand of DNA to serve as a template for the synthesis of the other strand. Base pairing also allows a strand of DNA to serve as a template for the synthesis of a complementary strand of RNA.  The DNA double helix o Two helical polynucleotide chains, coiled about a common axis. o The chains are antiparallel manner, the 5-end of one strand is paired with the 3- end of the other strand. o The hydrophilic deoxyribose-phosphate of each chain is on the outside of the molecule, while the hydrophobic bases are stacked on the inside. Base pairing o The bases of one strand of DNA are paired with the bases of the second strand so that an Adenine is always paired with a Thymine, while a Cytosine is always paired with a Guanine. Therefore one polynucleotide chain of the DNA double helix is always the complement of the other. Given the sequence of bases on one chain, the sequence of bases on the complementary chain can be determined. o The specific base pairing in DNA leads to Chargaff's rules: In any samples of duoble–strand DNA the amount of Adenine equals the amount of Thymine the amount of Guanine equals the amount of Cytosine and the total amount of purines (A + G) equals the total amount of pyrimidines (T + C). o The base pairs are held together by hydrogen bonds: two between A and T and three between G and C. These hydrogen bonds plus the van der Waals forces between the stacked bases stabilize the structure of the double helix.  DNA can occur in different three-Dimensional (3D) Forms DNA is a flexible molecule. Considerable rotation is possible around a number of bonds in the sugar–phosphate (phospho-deoxyribose) backbone. Many significant forms are described by Watson-Crick of DNA structure that are found in cellular DNA. These structural variations generally do not affect the key properties of DNA defined by Watson and Crick: strand complementarity, antiparallel strands, and the requirement for A=T and G = C base pairs.  The B form (DNA double helix)  Two helical polynucleotide chains, coiled about a common axis.  Right-handed  Chains are anti-parallel  Sugar-phosphates on the outside; bases on the inside.  Approx. 10 bases per helix turn and 0.34 nm between base pairs.  Rise/turn of helix = 3.57 nm Two structural variants that have been well characterized in crystal structures are the A and Z forms. These structural changes deepen the major groove while making the minor groove shallower. o The A form (Dehydration of DNA drives it into the A form).  Similar to the B form, but is more compact (0.26 nm between base pairs and 11 base pairs per turn).  Rise/turn of helix = 2.86 nm o The Z form  The bases of the two DNA strands are the left-handed helix.  0.37 nm between base pairs and 12 base pairs per turn.  Rise/turn of helix = 4.56 nm  DNA Supercoiling Supercoiling means the coiling of a coil. A telephone cord, for example, is typically a coiled wire. The path taken by the wire between the base of the phone and the receiver often includes one or more supercoils. DNA is coiled in the form of a double helix, with both strands of the DNA coiling around an axis. The further coiling of that axis upon itself produces DNA supercoiling. When there is no net bending of the DNA axis upon itself, the DNA is said to be in a relaxed state.  Alternative structures triplex and tetraplex (Quadruplex) Watson-Crick base pair, can form a number of additional hydrogen bonds. For example, Cytidine can pair with Guanosine (G=C), and Thymidine can pair with Adenosine (A=T). The N-7, O-6, and N-6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA, are often referred to as Hoogsteen positions, so called Hoogsteen pairing. Hoogsteen pairing allows the formation of triplex DNAs. Four DNA strands can also pair to form a tetraplex (quadruplex), but this occurs only for DNA sequences with Guanosine. The guanosine tetraplex, is stable over a wide range of conditions. Several types of RNA and RNA polymerases Messenger RNA (mRNA) template for protein synthesis RNA polymerase II (Pol II) Transfer RNA (tRNA) carries activated amino acids to ribosomes RNA polymerase III (Pol III) Ribosomal RNA (rRNA) major component of ribosomes RNA polymerase I (Pol I) All require template DNA, ribonucleotides (ATP, GTP, UTP, CTP) and a divalent metal ion (Mg2+) RNA DNA Biochemistry of Nucleic Acids (Gene Expression) Prepared by Dr. Amany M. Hamed Biochemistry Laboratory, Faculty of Science, Sohag University 2024-2025 Dr/ Amany Hamed DNA Replication Dr/ Amany Hamed Why DNA Replication ???? Fetal development and cell division Human development Human injury and wound healing Dr/ Amany Hamed Steps in DNA Replication Occurs when chromosomes duplicate (make copies) An exact copy of the DNA is produced with the aid of the enzyme DNA polymerase Hydrogen bonds between bases break and enzymes “unzip” the molecule Each old strand of nucleotides serves as a template for each new strand New nucleotides move into complementary positions are joined by DNA polymerase Dr/ Amany Hamed Steps in DNA Replication DNA replication starts at many points in eukaryotic chromosomes. DNA polymerases can find and correct errors. There are many origins of replication in eukaryotic chromosomes. Dr/ Amany Hamed Steps in DNA Replication Dr/ Amany Hamed Steps in DNA Replication Topoisomerase SSB (single strand binding protein) Dr/ Amany Hamed Steps in DNA Replication Primase Dr/ Amany Hamed Steps in DNA Replication DNA Polymerase III Dr/ Amany Hamed Steps in DNA Replication DNA Polymerase III and polymerase I Dr/ Amany Hamed Steps in DNA Replication Ligase Dr/ Amany Hamed Summary of Steps in DNA Replication Dr/ Amany Hamed Steps in DNA Replication Two New, Identical (semiconservative) DNA Strands Result from Replication Dr/ Amany Hamed Another View of Replication Dr/ Amany Hamed Proteins Dr/ Amany Hamed Protein Dr/ Amany Hamed Protein Dr/ Amany Hamed Protein Dr/ Amany Hamed Protein Dr/ Amany Hamed Protein Synthesis (Gene Expression) (Central Dogma) Dr/ Amany Hamed Dr/ Amany Hamed Ribosome Dr/ Amany Hamed Protein Synthesis DNA → RNA → Protein DNA Nuclear DNA membrane Transcription Transcription Pre-mRNA mRNA RNA Processing mRNA Ribosome Ribosome Translation Translation Protein Protein Prokaryotic Cell Eukaryotic Cell Dr/ Amany Hamed Dr/ Amany Hamed Dr/ Amany Hamed Protein Synthesis (Gene Expression) The production (synthesis) of polypeptide chains (proteins) consists of two phases: Transcription & Translation mRNA must be processed before it leaves the nucleus of eukaryotic cells. Dr/ Amany Hamed Making Proteins Step 1: Transcription Dr/ Amany Hamed The “Central Dogma” of Molecular Genetics Information passes from the genes (DNA) to an RNA copy of the gene, and the RNA copy directs the sequential assembly of a chain of amino acids Dr/ Amany Hamed The “Central Dogma” of Molecular Genetics RNA Polymerase Dr/ Amany Hamed The “Central Dogma” of Molecular Genetics Central Dogma: from Genes to Proteins Replication of the genes (DNA DNA) Transcribing the information (DNA RNA) Translating the nucleotide sequence into protein sequence (RNA Protein) – The Genetic Code – Protein Biosynthesis Dr/ Amany HamedThe “Central Dogma” of Molecular Genetics RNA is key to this process: Messenger RNA(mRNA): carries a copy of a DNA sequence to the site of protein synthesis at the ribosome Transfer RNA(tRNA): carries amino acids for polypeptide assembly Ribosomal RNA(rRNA): catalyzes peptide bond formation and provides the structure for the ribosome Dr/ Amany Hamed Making a Protein—Transcription First Step: Copying of genetic information from DNA to RNA called Transcription Why? DNA has the genetic code for the protein that needs to be made, but proteins are made by the ribosomes—ribosomes are outside the nucleus in the cytoplasm. DNA is too large to leave the nucleus (double-stranded), but RNA can leave the nucleus (single-stranded). Dr/ Amany Hamed Making a Protein—Transcription Part of DNA temporarily unzips and is used as a template to assemble complementary nucleotides into messenger RNA (mRNA). coding strand not transcribed Template strand transcribed Dr/ Amany Hamed Making a Protein—Transcription mRNA then goes through the pores of the nucleus with the DNA code and attaches to the ribosome. Dr/ Amany Hamed Steps in Transcription ❑ The transfer of information in the nucleus from a DNA molecule to an RNA molecule ❑ Only 1 DNA strand serves as the template ❑ Only one of the two strands of DNA, called the template strand, is transcribed. ❑ The strand of DNA that is not transcribed is called the coding strand. ❑ Starts at promoter DNA (TATA box) ❑ Ends at terminator DNA (stop) ❑ When complete, pre-RNA molecule is released Dr/ Amany Hamed What is the enzyme responsible for the production of the mRNA molecule? Dr/ Amany Hamed RNA Polymerase ▪Enzyme found in the nucleus ▪Separates the two DNA strands by breaking the hydrogen bonds between the bases ▪Then moves along one of the DNA strands and links RNA nucleotides together Dr/ Amany Hamed RNA Polymerase RNA polymerase unwinds DNA about ten base pairs at a time; reads template in 3’to 5’direction, synthesizes RNA in the 5’ to 3’ direction. The polymerase adds ribonucleotides to the growing 3′ end of an RNA chain. Bacteria contain only one RNA polymerase enzyme, while eukaryotes have three different RNA polymerases: 1.RNA polymerase I: synthesizes rRNA in the nucleolus. 2.RNA polymerase II: synthesizes mRNA. 3.RNA polymerase III: synthesizes tRNA. Dr/ Amany Hamed Processing Pre-mRNA Also occurs in the nucleus Pre-mRNA made up of segments called introns & exons Exons code for proteins, while introns do NOT! Introns spliced out by splicesome-enzyme and exons re-join End product is a mature RNA molecule that leaves the nucleus to the cytoplasm Dr/ Amany Hamed RNA Processing pre-mRNA molecule (TATA box) exon intron exon intron exon Splicing intron intron exon exon exon splicesome splicesome exon exon exon Mature mRNA molecule Dr/ Amany Hamed RNA Processing Cap 5` Poly tail 3` Guanine Adenine Dr/ Amany Hamed Messenger RNA (mRNA) Carries the information for a specific protein Made up of 500 to 1000 nucleotides long Sequence of 3 bases called codon AUG – methionine or start codon UAA, UAG, or UGA – stop codons  Dr/ Amany Hamed Messenger RNA (mRNA) start codon A U G G G C U C C A U C G G C G C A U A A mRNA codon 1 codon 2 codon 3 codon 4 codon 5 codon 6 codon 7 protein stop methionine glycine serine isoleucine glycine alanine codon Primary structure of a protein aa1 aa2 aa3 aa4 aa5 aa6 peptide bonds Dr/ Amany Hamed Making Proteins Step 2: Translation Dr/ Amany Hamed Dr/ Amany Hamed Making a Protein—Translation Second Step: Decoding of mRNA into a protein is called Translation mRNA (genetic code) Protein Transfer RNA (tRNA) carries amino acids from the cytoplasm to the ribosome. Dr/ Amany Hamed Making a Protein—Translation These amino acids come from the food we eat. Proteins we eat are broken down into individual amino acids and then simply rearranged into new proteins according to the needs and directions of our DNA. Dr/ Amany Hamed Making a Protein—Translation A series of three adjacent bases in an mRNA molecule codes for a specific amino acid—called a codon. Each tRNA has 3 nucleotides that are complementary to the codon in mRNA. Each tRNA codes for a different amino acid. Dr/ Amany Hamed Making a Protein—Translation mRNA carrying the DNA instructions and tRNA carrying amino acids meet in the ribosomes. Dr/ Amany Hamed Making a Protein—Translation Amino acids are joined together to make a protein. Dr/ Amany Hamed Making a Protein—Translation Dr/ Amany Hamed Making a Protein—Translation Use one of the codon charts on the next page to find the amino acid sequence coded for by the following mRNA strands. Dr/ Amany Hamed Making a Protein—Translation Dr/ Amany Hamed Making a Protein—Translation This table indicates the Genetic Code Dr/ Amany Hamed Transfer RNA (tRNA) Made up of 75 to 80 nucleotides long Picks up the appropriate amino acid floating in the cytoplasm Transports amino acids to the mRNA Have anticodons that are complementary to mRNA codons Recognizes the appropriate codons on the mRNA and bonds to them with H-bonds Dr/ Amany Hamed Transfer RNA (tRNA) amino acid attachment site methionine amino acid U A C anticodon Dr/ Amany Hamed Ribosomal RNA (rRNA) Made up of rRNA is 100 to 3000 nucleotides long Made inside the nucleus of a cell Associates with proteins to form ribosomes Dr/ Amany Hamed Ribosomes Made of a large Large and small subunit subunit Composed of rRNA P A (40%) and proteins Site Site (60%) Have two sites for tRNA attachment mRNA --- P and A Small A U G C U A C U U C G subunit Dr/ Amany Hamed Translation Synthesis of proteins in the cytoplasm Involves the following: 1. mRNA (codons) 2. tRNA (anticodons) 3. ribosomes 4. amino acids Dr/ Amany Hamed Steps of Translation Large subunit P A mRNA Codons Join Site Site the Ribosome mRNA Small A U G C U A C U U C G subunit Dr/ Amany Hamed Steps of Translation Initiation Dr/ Amany Hamed Steps of Translation Elongation Dr/ Amany Hamed Steps of Translation Dr/ Amany Hamed Steps of Translation Dr/ Amany Hamed Steps of Translation Dr/ Amany Hamed Steps of Translation Dr/ Amany Hamed Steps of Translation Dr/ Amany Hamed Steps of Translation Termination Dr/ Amany Hamed End Product –The Protein! The end products of protein synthesis is a primary structure of a protein A sequence of amino acid bonded together by peptide bonds aa5 aa4 aa3 aa2 aa199 aa1 aa200 Dr/ Amany Hamed Protein Synthesis (Gene Expression) Translation Video Protein Synthesis Video