630 Microbiology forTeachers

Principles of Molecular Biology

Definitions

• Macromolecules
  • large molecules, typically comprised of a number of subunits attached to one another to form polymers
  • examples include
    • nucleic acids (polynucleotides) composed of nucleotides (which have three components ... nitrogenous base, sugar, phosphate) linked by phosphodiester bonds
      • DNA is composed of four nucleotides and can be:
        • single-stranded, but can form secondary structures based on short sequences of hydrogen bond interactions between nucleotides
        • double-stranded ... based on hydrogen bond interactions between nucleotides to form a double-helix
      • RNA can be:
        • single-stranded, but can form secondary structures based on short sequences of hydrogen bond interactions between nucleotides
        • double-stranded ... based on hydrogen bond interactions between nucleotides
    • proteins (polypeptides) composed of amino acids
      • primary structure = sequence of amino acids in polymers
      • secondary structure = two-dimensional arrangement of amino acid groups into alpha-helices and beta-pleated sheets, stabilized by hydrogen bonding
      • tertiary structure = three-dimensional conformation of polypeptides
    • polysaccharides
    • lipids ... two major types
      • glycerides (mono-, di-, and triglycerides)
        • glycerol backbone
        • fatty acids attached to glycerol (via ester bonds or ether bonds)
      • steroids
  • large molecules problem set website ... test your understanding!
  • macromolecules are the building blocks of cells
    • DNA in cells
• Genetics
  • study of heredity, which is determined by genes
  • genes are informational molecules (typically DNA) which code for the production of either a polypeptide or RNA
• Central dogma - the concept that biological information is always encoded in nucleic acid molecules
  • DNA is the main repository of genetic information (some viruses do store their genetic information in the form of RNA, however)
  • RNA is transcribed (or copied) from DNA whenever it is necessary to utilize the genetic information, such as in synthesis of proteins, rRNA or tRNA
  • protein is synthesized by translation of the information encoded in triplets of mRNA bases called codons, into the sequence of amino acids in the specific protein

Replication

Replication is the duplication of informational molecules ... in this case DNA

• Strand separation - catalyzed by helicase
• Single-strand maintenance - binding of single strand binding protein
• Primer formation - catalyzed by primase, which generates an RNA primer that helps initiate synthesis at the origin of replication
• Base pairing - binding (via hydrogen bonds) of complementary nucleotides to bases in the strand to be copied
• Polymerization - catalyzed by DNA polymerase III, which generates short fragments
• Primer removal - catalyzed by DNA polymerase I, which also completes the fragments by replacing the RNA primer with appropriate DNA sequence
• Ligation - DNA fragments are attached to one another to form a continuous strand
• Proofreading - catalyzed by DNA polymerase III which removes improperly base-paired nucleotides and replaces them with the correct nucleotide
• Double-strand formation - when replication enzymes dissociate, the helix reforms by base pairing

This mode of replication is referred to as semi-conservative

Transcription

Transription is the generation of an mRNA copy of the genetic information coded in the DNA

• Strand separation - catalyzed by the "core enzyme" of RNA polymerase
• Promoter recognition - sigma factor binds to the promoter sequences, which are generally comprised of the -35 sequence (consensus is TTGACA) and the -10 sequence (Pribnow box, consensus is TATAAT)
• Base-pairing - binding (via hydrogen bonds) of complementary nucleotides to bases in the strand to be copied
• Polymerization - this is catalyzed by RNA polymerase starting at the so-called start codon, which is generally adenine (sigma factor is released before this begins)
• Termination - reading of the DNA code stops in one of two ways:
  • at termination sequences, which generally consist of sequences that result in formation of stem-loop structures
  • rho binding, which causes RNA polymerase to dissociate from the DNA

Translation

Translation is the mRNA-mediated process by which proteins are generated based on amino acid sequence information coded in the DNA

• Initiation - binding of the Shine-Dalgarno sequence by 16S RNA of the ribosome allows firm attachment of the mRNA to the small subunit (30S) of the ribosome, which then binds several initiation factors and the large subunit (50S) of the ribosome, which binds formylmethionine tRNA (bound to the mRNA start codon, AUG) at the peptide site (P site)
• Elongation
  • the next codon is recognized by the anticodon of the corresponding tRNA, which binds at the acceptor site (A site)
  • next, a peptide bond is formed between adjacent amino acids (the enzyme used is a ribozyme ... i.e., it is a catalytic RNA) assisted by elongation factors, and the growing peptide is retained bound to the amino acid (still attached to its tRNA that is attached to the A site)
  • the tRNA molecule which was in the P site moves to the exit site (E site), then dissociates, and the tRNA-peptide complex translocates (energy provided by GTP) to the P site
• Termination-release - when a codon is reached which does not specify a tRNA-amino acid complex; stop or nonsense codons (UAA, UAG, UGA) release factors cleave the growing peptide from the last tRNA, then the ribosome dissociates into its 30S and 50S components
• Folding - based on its amino acid sequence, the nascent polypeptide folds itself into its most stable conformation as it is being generated; it may be assisted by chaperonins when folding