Biochemistry

• Replication


  Semiconservative process where each strand of DNA serves as a template for a new strand, resulting in genetic information being faithfully copied into a new generation

  
• Replication

  • Bidirectional - begins at origin(s) and proceeds in both directions
  • Semidiscontinuous - leading strand and lagging strand with Okazaki fragments
• Components of replication protein


  • DNA helicase
  • DNA gyrase (topoisomerase)
  • SSB (single-stranded DNA binding)
  • DnaA (initiation factor)
  • HU (histone-like)
  • Pri A, B, C (primosome assembly)
  • Dna B, C and T (DNA unwinding)
  • Primase (RNA primer synthesis)
  • DNA polymerase III holoenzyme (elongation)
  • DNA polymerase I (excises RNA primer, fills in with DNA)
  • DNA ligase (links Okazaki fragments)
  • Tus (termination)

  
• Steps of replication


  1. DNA unwinding and initiation

  2. DNA priming

  3. DNA elongation and extension

  4. Termination

  
• DNA polymerases

  Polymerize new DNA or RNA against existing template in replication and transcription

  
• DNA polymerase I


  • Proofreads and corrects typos, repairs mismatched bases, removes abnormal bases, reduces error rate from 1 in 10,000 to 1 in 100 million bases

  
• Telomerase

  Enzyme that adds DNA sequence repeats to the 3' end of DNA strands in telomere regions to maintain telomere length

  
• Differences between prokaryotic and eukaryotic replication

  • Prokaryotes have one origin, unwinding is bidirectional
  • Eukaryotes have multiple origins, DNA polymerases are main enzymes with proofreading, telomeres cap linear chromosomes
• Chemotherapeutic anti-cancer agents and fluoroquinolone antibiotics disrupt DNA replication by targeting topoisomerases

  
• Transcription

  Synthesis of RNA molecule using DNA as a template, transferring information from DNA to single-stranded RNA for protein synthesis

  
• Cellular RNAs


  • Messenger RNA (mRNA)
  • Ribosomal RNA (rRNA)
  • Transfer RNA (tRNA)
  • Small nuclear RNAs (snRNAs)
• Transcription components

  • Template (DNA strand)
  • Substrate (ribonucleoside triphosphates)
  • Enzyme (RNA polymerase)

  
• Prokaryotic RNA polymerase


  Single enzyme that transcribes all three RNAs, contains core enzyme and sigma factor
• Eukaryotic RNA polymerases

  • RNA polymerase I (rRNA)
  • RNA polymerase II (mRNA, snRNA)
  • RNA polymerase III (tRNA, 5S rRNA)
• Stages of transcription


  1. Initiation

  2. Elongation

  3. Termination
• Prokaryotic promoters


  • Pribnow box (-10 region)TATAAT, -35 regionTTGACA
• Eukaryotic promoters


  • TATA box, CAAT box, enhancer sequences
• Enhancers


  DNA sequences that stimulate transcription frequency, can be upstream, downstream or within genes
• Silencers


  DNA sequences that bind proteins to inhibit transcription rate

  
• Compounds that inhibit RNA synthesis include actinomycin D, rifampicin, and α-amanitin
• Enhancers


  DNA sequences that regulate the frequency of transcription of genes in eukaryotic cells. They can stimulate the transcription of genes.
• Location of enhancers

  Can be upstream, downstream, or within genes
• Silencers

  DNA sequences which bind proteins that act to inhibit the rate of transcription

  
• A particular enhancer is effective only in certain cells, e.g. the immunoglobulin enhancer function in B-lymphocytes but not elsewhere
• Cancer can result if the relation between genes and enhancer is disrupted

  e.g. a chromosomal translocation brings the proto-oncogene myc under the control of immunoglobulin enhancer which leads to dysregulation of the myc gene and is believed to play role in development of cancer, leukemia and lymphoma
• Inhibitors of RNA synthesis

  Compounds that inhibit RNA synthesis by binding to DNA or RNA polymerase

  
• Inhibitors of RNA synthesis in prokaryotes but not eukaryotes

  • Rifampin
  • α-Amanitin
  • Dactinomycin (actinomycin D)
• Rifampin

  Anti-tuberculosis drug that inhibits the initiation of transcription by binding β-subunit of RNA polymerase. Has no effect on eukaryotic nuclear RNA (nRNA) polymerases.

  
• α-Amanitin

  Compound derived from the poisonous mushroom Amanita Phalloides that inhibits eukaryotic RNA polymerase
• Dactinomycin (actinomycin D)

  Therapeutic agent in the treatment of some cancers. Binds tightly and specifically to double helical DNA and thereby prevents it from being an effective template for RNA synthesis. At low concentrations, inhibits transcription without affecting DNA replication or translation. Used as an inhibitor for the formation of new RNA in both prokaryotes and eukaryotes. Its ability to inhibit the growth of rapidly dividing cells makes it an effective therapeutic agent in the treatment of cancers.

  
• Post-transcriptional processing

  • Cleavage of large precursor of RNA
  • Splicing
  • Terminal addition of nucleotides
  • Nucleoside modification
• Eukaryotic tRNA processing

  • Cleavage of 5' leader sequence
  • Splicing to remove intron
  • Replacement of 3' terminal UU by CCA
  • Modification of several bases
• Eukaryotic rRNA processing

  Three of the eukaryotic rRNAs (28S, 18S and 5.8S) are synthesized from long precursor called preribosomal (45S) RNAs. The fourth, 5S rRNA is produced by the transcription of 5S gene by RNA polymerase lll and is modified separately. The 45S rRNA precursors are cleaved and trimmed to produce the mature function rRNA species. Spacer sequences are removed by cleavage of 45S RNA by specific endonucleases. The 5.8S RNA base pairs with the 28S rRNA during formation of the ribosomal subunits, which is completed before transport from the nucleus.

  
• Eukaryotic mRNA processing

  Mature, functional mRNA is formed from processing of a large precursor called hnRNA (heterogenerous nuclear RNA) transcript product of RNA polymerase ll. Includes 5' Capping, Addition of poly (A) tail, and Splicing.

  
• 5' Capping

  The 5' end of eukaryotic mRNA consists of cap of 7-methylguanosine triphosphate. The addition of the guanosine triphosphate is catalyzed by the nuclear enzyme guanyltransferase and Methylation is catalyzed by guanine-7-methyl transferase.

  
• Addition of poly-A tail

  Eukaryotic primary transcript are cleaved by a specific endonuclease that recognizes the sequence AAUAA. A poly (A) polymerase adds about 200 to 300 adenylate residues to the 3' end of the transcript, ATP is the donor of adenylate residues.

  
• Splicing


  The process of removing introns and joining exons. The molecular complex that accomplishes these tasks is known as the spliceosome (several small RNAs and proteins). All introns begin with a 5' GU and end with a 3' AG.
• snRNP (snurps) involved in splicing

  • U1 - Binds the 5'splice site and then 3' splice site
  • U2 - Binds the branch site of the introns
  • U4 - Masks the catalytic activity of U6
  • U5 - Binds the 5' splice site
  • U6 - Catalyzes splicing

  
• Translation


  The process by which ribosomes convert the information carried by mRNA in the form of genetic code to the synthesis of new protein. It occurs in the cytosol.

  
• Genetic code

  The system of nucleotide sequences of mRNA that designates particular amino acid sequences in the process of translation. The codons are written in the 5' to 3' direction.