Nucleic Acid Structure: Purine and Pyrimidine nucleotides can be combined to form nucleic acids: 1. Deoxyribonucliec acid (DNA) is composed of deoxyribonucleosides of! Adenine! Guanine! Cytosine! Thymine 2. Ribonucleic acid (RNA) is composed of ribonucleoside of! Adenine! Guanine! Cytosine! Uracil Purines and pyrimidines are critical bc of their use in:! the synthesis of ATP! cofactors! RNA! DNA and other important cell components Nearly all mos can synthesize their own purines and pyrimidines they are critical to cell function. Purines and Pyrimidines are cyclic nitrogenous bases with several double bonds and aromatic properties. Purines:! Two joined rings! Adenine and guanine are commonly found in MOs Pyrimidines:! One ring! Uracil, cytosine and thymine are commonly found in MOs A purine or pyrimidine base joined with a pentose sugar, either ribode or deoxdyribose is a nucleoside.
Nucleotide: is a nucleoside with one or more phosphate groups attached to a sugar.! In both DNA and RNA, nucleosides are joined by phosphate groups to form long polynucleotodes chains.! Difference in chemical composition is the: Sugar and pyrimidines bases: DNA: deoxyribose and thymine RNA: ribose and uracil in place of thymine Structure and function of DNA: 1. Bacterial chromosomes consist of single circular molecules of doublestranded DNA (dsdna). a. Size of the E. coli chromosome is 4.7 x 10 6 base pairs (bp). b. Size of an average bacterial gene is 1,000 bp (E. coli has about 3,500 genes vs. human 30,000). 2. Structure of DNA a. Two polynucleotide chains that contain the bases adenine (A), cytosine (C), guanine (G), and thymine (T). b. DNA is a double helix consisting of complementary strands the bases in one strand match up with those of other according to the base pair rules.! purine Adenine (A) is always paired with the pyrimidine thymine (T)! A = T! Purine guanine (G) is always paired with the pyrimidine Cytosine.! G = C, hydrogen bonds broken by heat). c. Two properties of the genetic material. (1) The genetic material undergoes replication prior to cell division.
(2) The genetic material directs protein synthesis. d. Important concept: a single nucleic acid strand (usually DNA) can be used to guide the synthesis of a complementary strand (either DNA in replication or RNA prior to protein synthesis). The original strand is a template for complementary strand synthesis. 3. RNA Structure:! Usually single stranded! Can coil back upon itself to form a hairpin-shaped structure with complementary base pairing and helical organization! Cells contain three (3) different types of RNA: 1. messenger RNA mrna 2. ribosomal RNA rrna 3. transfer RNA trna (differ in function,, site of synthesis in ec, and structure) DNA replication: 1. Bidirectional Replication a. Replication starts at a single site on the circular DNA E. coli chromosome (origin of replication). b. Replication stops at a site about half-way around the chromosome (the terminus of replication). c. Replication forks are the sites of new DNA synthesis, the place where the DNA helix is unwound and individual strands are replicated. d. Replication of the E. coli chromosome is bidirectional (two replication forks moving in opposite directions around the chromosome). e. Replication continues until the entire replicon is replicated
! Replicon: portion of the genome that contains an orgin and is replicated as a unit.! And when the replication forks move around the circle, the bacterial chromosome is a single replicon, the forks meet on the other side and two separate chromosomes are released. 2. Rolling Circle Replication! Occurs during E.coli conjugation and reproduction of viruses. a. One strand of DNA is nicked and the free 3 hydroxyl end is extended by replication enzymes. b. As 3 end is lengthened while the growing point rolls around the circular template, the 5 end of strand is displaced and forms an everlengthening tail. c. The single stranded tail may be converted to the double stranded form by complementary strand synthesis. d. Useful for viruses bc allows rapid and continuous production of many genomes copies from a single initiation event. Mechanism of DNA Replication: (1) The double helix is unwound by DNA helicase (requires ATP for activity). (2) Single-strand DNA binding protein (SSBs) binds to and stabilizes the unwound DNA strands. (3) During the process of rapid unwinding the DNA, tension, supercoils and supertwists can occur in the helix, tension relieved and continued unwinding is promoted by the a topoisomerase.! Topoisomerase changes the structure of DNA by breaking one or two strands that it remains unaltered as its shape is changed.! DNA gyrase is an E.coli topooisomerase.
(3) DNA polymerase III requires a primer (a free 3' end onto which new nucleotides can be attached). Therefore, short RNA primers are first synthesized by primase.! DNA polymerase enzymes: catalyzes the synthesis of DNA in the 5 to 3 direction and reading the DNA template in the 3 to 5 direction. (4) DNA polymerase III synthesizes the new complementary DNA strands using each parental strand as a template. (a) Synthesis is continuous on one strand and discontinuous on the other. - DNA replication always proceed from the 5 phosphate to the 3 hydroxyl. - Leading Strand: strand growing from the 5 phosphate to the 3 hydroxyl. - DNA synthesis can occur continuously bc there is always a free 3 OH at the replication fork to which a new nucleotide can be added. - Lagging Strand: the opposite strand, DNA synthesis must occur discontinuously (bc there is no 3 OH at the replication fork to which a new nucleotide can attach.) (b) Discontinuous fragments (Okazaki fragments) are about 1,000 nucleotides in length. (5) DNA polymerase I removes the RNA primers and replaces them with DNA (repair enzyme). (6) DNA ligase joins and seals the pieces of newly-synthesized DNA together. f. The enzymes involved in DNA replication form a complex (the replisome) at each replication fork. g. Both of the daughter chromosomes consist of one parental DNA strand and one newly-synthesized DNA strand (semi-conservative replication).
Expression of the genetic information a. The sequence of bases along the double helix can be read by cell machinery and used as a blueprint to make proteins (the genetic code Table11.5).! DNA base sequence corresponds to the amino acid sequence of the polypeptide specified by the gene.! Mutations are the results of changes of single amino acids in a polypeptide chain.! There are 20 amino acids present in a protein, therefore there must be 20 different code words in a linear single strand of DNA.! Codons: code words, a sequence of three bases in messenger RNA that encodes for specific amino acid. 1. Code degeneracy: there are up to six different codons for a given amino acid. 2. 61 codons, Sense codons direct amino acid incorporation into protein. 3. Stop or Nonsense codons: the remaining 3 (UGA, UAG, UAA) are involved in the termination of translation. b. The genetic information encoded in DNA directs protein synthesis in two steps: transcription and translation. DNA Transcription or RNA Synthesis: Transcription: synthesis of RNA under the direction of DNA! Generates three (3) KINDS OF RNAs (1). Messenger (mrna): o bears the message for protein synthesis (2). Transfer (trna): o carries amino acids during protein synthesis (3). Ribosomal (rrna): o molecules are components of ribosomes
(1) Gene: a sequence of bases in DNA that specifies a single polypeptide (or, in some cases, a single RNA molecule rrna, trna). (2) Organization of a typical gene in E. coli: promoter - coding region - terminator. (3) RNA polymerase enzyme makes a single-stranded RNA copy (messenger RNA) that is complementary to one of the DNA strands (the template strand) of the gene, therefore mrna is synthesized under the direction of DNA.! Like DNA, RNA synthesis processed in the 5 to 3 direction with new nucleotides being added to the 3 end of the growing chain.! There are two enzymes in E.coli that help aid in the transcription process: 1. Core Enzyme: helps by catalytic RNA synthesis and contains four types of polypeptide chain. 2. Sigma Factor Enzyme: has no catalytic activity but helps the core enzyme recognize the start genes. Once RNA synthesis begins, the sigma factor dissociates from the core enzyme-dna complex and is available to aid another core enzyme (a) (b) RNA polymerase recognizes a specific base sequence with the aid of the sigma factor and binds to the promoter, where it unwinds the DNA strands (16 base pairs) and begins mrna synthesis (on one of the DNA strands - the coding strand). Enzyme continues ATP-dependent mrna synthesis as it moves through the coding region.
(c) Enzyme stops at the terminator and releases both mrna and DNA. Translation or Protein Synthesis:! Terminators: stop signals to mark the end of a gene or sequence of genes and stop transcription by the RNA polymerase.! Two kinds of terminators: 1. Stretch of six uridine residues following the mrna and causes the polymerase to stop transcription and release the mrna without the aid of any accessory factors. 2. Rho Factor: Special protein It is thought that the rho binds to mrna and moves alongthe molecule until it reaches the RNA polymerase that has halted at a terminator. Rho then causes the polymerase to dissociate from the mrma, probably by unwinding the mrna-dna complex. Translation: mrna nucleotide sequence is translated into the amino acid sequence of a polypeptide chain. (1) Each set of three bases in mrna (a codon) specifies one amino acid in a protein. (2) The dictionary of codons constitutes the genetic code (3) Amino acids do not line up directly with the codons: an adapter molecule called transfer RNA is required. (a) The amino acid is attached to the 3 end of the trna molecule.! The 3 end of all trnas has the same C-C-A sequence.
(b) (c). (d). Other end of the trna molecule carries a triplet of bases (the anticodon) that is complementary to the mrna codon. The overall structure of the trna is a cloverleaf structure p. 266 Amino acids are activated for protein synthesis through a reaction catalyzed by aminoacyl-trna synthetases AA + trna + ATP# aminoacyl-trna = AMP + ppi Process where the amino acid is activated: -- amino acid is attached to the 3 OH of the trna and readily transferred to the end of the growing polypeptide chain. (4) Amino acid-charged trna s and mrna are brought together at a complex cellular organelle called a ribosome.! The place where protein synthesis actually takes place The ribosome consists of two subunits. (a) (b) small (30S) subunit: 1 ribosomal RNA molecule and 21 proteins large (50S) subunit: 2 ribosomal RNA molecules and 32 proteins
Steps in Translation (Protein Synthesis) (a) Initiation of polypeptide synthesis when mrna, small ribosomal subunit, first charged trna, and accessory initiation factors bind together (initiator codon AUG or GUG). Binding of the large ribosomal subunit follows.! Bacteria begin translation with a N-formymethionyltRNA fmet! Eucaryotic and Archeal (except mitochindrian and chloroplast) begin translation with a special initiator methinoyl-trna Met (b) Elongation of the polypeptide occurs as new trna's are brought in and old trna's are expelled from the ribosome following peptide bond synthesis. [1] Initial entry of charged trna and peptide bond formation occur in the A site.! Aminoacyl or acceptor site [2] Expulsion of preceding (now uncharged) trna from the P site.! Peptidyl or donor site [3] Translocation: the final stage in elongation. Three things happen here:! Transfer of trna-linked polypeptide moves from the A site into the P site.! Ribosomes moves one codon along the mrna so tha a new codon is positioned in the A site.! Empty trna leaves the P site t the E site and then leaves the ribosome.
(c) Release or Termination of polypeptide and mrna from the ribosome upon the completion of translation.! Small and large ribosomal subunits dissociate with the aid of accessory release factors.! Protein synthesis stops when it reaches one of the nonsense codons: UAA, UAG, and UGA.! Release Factors(RF-1, RF-2, RF-3). aids the ribosome in recognizing the nonsense codons. 5. Regulation of gene expression a. Expression of most genes can be turned off and on, usually by controlling the initiation of transcription. b. Lactose degradation in E. coli (1) LacY protein transports lactose into the cell. (2) LacZ protein (β-galactosidase) cleaves lactose (a disaccharide) into glucose and galactose. (3) LacA protein is a nonessential enzyme. c. Operon: several genes are transcribed as a single unit, usually encode proteins involved in a common process (not common in eukaryotes). d. Organization of the lac operon
e. When lactose is added to the culture medium, transcription of the lac operon is induced. In the absence of lactose, transcription is repressed. f. Regulation is mediated by the lactose repressor protein which has a (1) lactose binding site (2) DNA binding site (specific for operator sequence) g. When lactose is absent, repressor binds to DNA at the lac operator and prevents RNA polymerase from binding to the lac promoter. Transcription is blocked. h. When lactose is present, repressor binds to it and is then unable to bind to DNA. RNA polymerase can now bind to lac promoter and transcription begins.