CHAPTER 34

• TRANSFER RNA

  • Make up 15-20% of cellular RNA

  • Smallest of the 3 major species of RNA

  molecules

  • Serve as “adaptor molecule” that carries its specific amino acid to the site of protein synthesis

  • 20 species of tRNA à at least 1 specific type of tRNA molecule for each of the 20 amino acids

  • Single stranded but have extensive folding because of intra-strand complementarity

  • Assumes a cloverleaf-like structure (secondary level of structure
• 5 loops/arms of TRANSFER rna

  1. Dihydrouracil arm
  2. Anticodon arm
  3. Extra arm
  4. Pseudouracil thymidine arm
  5. Acceptor arm
• TRNA

  the 3’ end always end in CCA sequence

  • Amino acid is always attached to the ‘A’ at the 3’ end
• RIBOSOMAL RNA

  • Make up 80% of the cellular RNA

  • 2 major nucleoprotein subunits

  • Large 60s – catalyzes the peptide bond

  • Small 40s –decodes the genetic message

  • Acts as the translation machinery for the synthesis of proteins from the mRNA
• • SnRNAs (small nuclear): mRNA & tRNA processing and gene regulation
  • Small ncRNAs (small noncoding): miRNA & siRNAs inhibit gene expression; potential agents for therapeutic drug development
  • Large ncRNAs (large noncoding): lncRNAs & circRNA contribute to structure of chromatin regulation of gene expression
• SMALL RNA

  • Make up <5% of cellular RNA

  • Not directly involved in protein synthesis
• NUCLEASES

  • Enzymes that are capable of degrading nucleic acid phosphodiester bonds

  • Specificities:

  • Hydrolyze both DNA and RNA

  • Hydrolyze only DNA(deoxyribonucleases)or RNA (ribonucleases)

  • Hydrolyze single and double stranded nucleic acids
• Exonucleases – capable of hydrolyzing nucleotides at the terminal of a molecule

  • Endonuclease – within the strand

  • Restriction endonucleases recognize specific sequences in DNA and cleaving both strand

  • CRISPR-Cas family of enzymes uses a guide RNA of specific nucleotide sequence that targets a nuclease to cleave distinct DNA or RNA sequences
• MESSENGER RNA

  • Make up 2-5% of cellular RNA

  • Carry the genetic information from the DNA to the cytosol, where it is used as a template for protein synthesis

  • Monocistronic – carries only the information for the production of a single polypeptide
• MESSENGER RNA

  • Unique chemical characteristics has a methylguanosine cap at the 5’ end and a polyadenine tail at the 3” end
• MESSENGER RNA

  • 5’ end cap serve as recognition site by the translation machinery and protection from attack of 5’ exonucleases

  • Translation starts at the 5’ end of the mRNA
• • 3’ hydroxyl terminus poly-A tail protects mRNA from the attack of 3’ exonucleases
• NUCLEOTIDES ABSORB ULTRAVIOLET LIGHT

  • UV light is absorbed within the conjugate double bonds of purines and pyrimidines

  • chemical modification effect of UV light on the double bonds cause its mutagenicity on the DNA
• Renaturation refers to the process where denatured, complementary strands of DNA can reform a duplex DNA structure
• Reannealing process is often referred to as hybridization

  • Hypochromic effect – absorbance decreases

  • Should be below Tm with high salt

  concentration

  • Rate of annealing/hybridization depends on the concentration of the complementary strands
• Hyperchromicity of denaturation the optical absorbance of purine and pyrimidine bases increases upon denaturation
• The melting temperature (Tm) is the temp at which half the DNA is denatured

  Base composition affects the Tm - more GC base pairs in DNA, the higher is its Tm
• Higher salt concentration - can shield the interchain repulsion of negatively charged phosphates of the phosphodiester bonds à higher Tm
• Denaturation happens when:

  • HIGH Melting temperature • LOW Salt concentration

  • FORMAMIDE – solvent used in recombinant DNA

  • Destabilizes the hydrogen bonds à decreasing Tm that denatures DNA à minimizing phosphodiester bond breakage and chemical damage to nucleotides
• DENATURATION (DISSOCIATION) OF DNA

  • When duplex DNA molecules hydrogen bonds between base pairs are disrupted, the strands are no longer held together

  • The double helix is denatured
• DNA

  • Serves as the genetic material in most organisms

  • Stores information to direct and regulate construction of proteins

  • Directs RNA synthesis

  • Directs protein synthesis through RNA
• FORCES THAT STABILIZE NUCLEIC ACID DOUBLE HELIX

  Two major forces contributing to the stability of the helix:

  1. Hydrogen bond between base pairs
  2. Van der Waals and hydrophobic interactions in base stacking
• THE WATSON – CRICK MODEL OF DNA

  • Two strands are antiparallel • Right-handed double helix

  • Chargaff’s rules

  • Watson-Crick base pairing restriction • dGTP and dCTP

  • dATP and TTP
• NUCLEIC ACIDS

  • Nucleic acids are responsible for the storage and passage of the information needed for the translation of proteins

  • Polymers of nucleotides

  • Held together by:

  • Bound between adjacent sugar moiety
• NUCLEOTIDES

  NUCLEOSIDE + PO4

  n One or more phosphate groups esterified to the sugar via a phosphoester bond
• NUCLEOSIDES

  BASE + SUGAR

  n 2’-deoxyribose

  n N-glycosidic linkage between C-1’ of the sugar and N-9 (purine) or N-1 (pyrimidine)
• monomeric deoxynucleotide units of DNA— deoxyadenylate, deoxyguanylate, deoxycytidylate, and thymidylate