Lec. 1

  1. Karyotype
    Normal complement of chromosomes
  2. Telomeres
    the ends of linear chromosomes; specialized protein-DNA structures that contain long stretches of the hexameric repeat TTAGGG
  3. Telomere Replication
    • - with every generation a short section of DNA is lost (telomere attrition or telomere erosion) because there is no way to replace the RNA primer with DNA since there is no 3' nucleophilic OH group
    • - when the telomeres reach a critical minimal length (the Hayflick limit) cells will senesce
  4. 5'-TTAGGG-3'
    this sequence is non-coding and acts as a buffer against telomere attrition (degradation)
  5. Telomerase
    • -is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG
    • -consists of two subunits each of TERT and TERC
    • -not active in the vast majority of adult somatic cells, but is active in germ cells, stem cells of self-renewing tissues, and is re-activated in a high percentage of cancer cells
  6. TERT

    Telomerase Reverse Transcriptase (can synthesize single-stranded DNA from single-stranded RNA in a reverse transcription process (RNA-dependent DNA polymerase)

    Telomerase RNA
  7. 6 different types of RNA
    tRNA, rRNA, mRNA, snRNA, siRNA, miRNA
  8. Messenger RNA (mRNA)
    • -least abundant
    • -sequences of the bases specify the order of amino acids in proteins
    • -heterogeneous in length
    • -highest turnover (least stable) of most RNAs, and the nucleotides can be recycled
  9. RNA polymerase (E.Coli)
    • - multisubunit enzyme termed the holoenzyme
    • - sigma subunit is weakly attached and is involved in the recognition of promoter sequences
    • - the promoter is the DNA sequence that signals the start of transcription
    • - alpha2omegabetabeta' is the core enzyme and comprises the active polymerization unit (lacks specificity)
  10. Overview of Prokaryotic Transcription
    • -RNA pol reads the 3'-5' strand (template or antisense strand) and synthesizes RNA in the 5'-3' direction
    • -top strand is the coding or sense strand
    • -the coding strand has the same sequence as the RNA that is produced (except U replaces T)
  11. Promoter
    • -specific DNA sequence that marks the start of a gene
    • -sigma- subunit of RNA pol recognizes the promoter sequence
    • -typically AT-rich, more easily unwound
    • -the stronger the promoter, the tighter RNA Pol binds, the greater the frequency of gene transcription, more mRNA, more protein produced
  12. 4 important promoter elements
    1. +1 transcription start site

    2. -10 region (Pribnow box)

    3. -35 region (or -35 element)

    4. UP element (-40 to -60 and enhances binding)
  13. Consensus Sequence
    a DNA or RNA sequence that is conserved (or very similar)

    -eg. TATAAT for the Pribnow box
  14. Transcription--Chain Initiation
    1. begins when RNA Pol binds to the promoter and forms the 'closed complex'

    2. the sigma subunit recognizes the promoter and facilitates the binding of RNA to the promoter (confers specificity)

    3. bridges the -10 and -35 regions to the RNA Pol via a flexible flap in the sigma subunit

    4. requires the formation of an open complex

    5. beta' and sigma subunits initiate strand separation (helicase activity)

    6. a purine ribonucleoside triphosphate is the first base in RNA (typically A)

    7. first residue retains its 5'triphosphate (PPP)

    8. no primer is necessary
  15. Transcription-Chain Elongation
    1. after strand separation, a transcription bubble moves along the DNA to be transccribed

    2. RNA Pol catalyzes the formation of a phosphodiester bond between ribonucleotides

    3. after about 10 nucleotides have been incorporated, the sigma subunit dissociates and is recycled which increases the speed and processivity of RNA Pol

    4. the rate is not constant, and is dependent on the DNA sequence and tertiary structure
  16. 2 types of Transcription--Termination
    Type I: Intrinsic

    Type II: Rho dependent termination
  17. Intrinsic Termination
    1. controlled by specific sequences called termination sites, characterized by two inverted repeats separated by thymines

    2. inverted repeats are sequences of bases that are complementary

    3. When the RNA is produced it can loop back on itself to form a hairpin loop

    4. the haripin loop stalls the RNA Pol

    5. the presence of the Us also causes a series of weak A-U base pairs between the template strand and the RNA, additionally causing the RNA Pol to dissociate
  18. Rho-dependent termination
    1. based on the formation of an RNA hairpin loop

    2. it involves the activityof a helicase protein called rho

    3. the rho protein binds to the RNA and follows RNA Pol during transcription

    4. when the RNA Pol transcribes the inverted hairpin loop sequence it stalls

    5. the rho protein 'catches-up' and facilitates the dissociation of the RNA Pol

    6. rho dependent termination is an ATP dependent process
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Lec. 1