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2011-11-28 19:27:22
Genetics Chapter 14

RNA Molecules and RNA Processing
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  1. Structure of Prokaryotic mRNA
    Shine-Dalgarno sequence: the site where ribosomes bind to the mRNA; 7 nts upstream of the start codon
  2. Coding Region
    • Specifies the amino acid sequence
    • -i.e. where the codons are found
  3. Key Concept
    Prokaryotic mRNA CAN contain more than 1 coding region

    a polycistronic ('multigenic') RNA

    one promoter -> mRNA -> multiple polypeptides
  4. Key Concept
    Eukaryotes have mostly monocistronic RNAs
  5. Eukaryotic Transcription
    • 1) Involves MORE enzymes & factors
    • 2) Occurs even though the DNA is packaged into NUCLEOSOMES
    • 3) Is physically SEPARATE from all translation activities
  6. Three RNA Polymerases in Eukaryotes
    RNA Polymerase I (Large rRNAs), RNA Polymerase II (pre-mRNA, some snRNAs, snoRNAs), RNA Polymerase III (tRNAs, small RNA, some snRNAs)
  7. Key Concept
    RNA Polymerase II is responsible for txn of MOST eukaryotic genes
  8. THREE Stages of Transcription
    • 1) Initiation
    • 2) Elongation
    • 3) Termination
  9. Key Concept
    Eukaryotic promoters can consist of MANY RECOGITION SEQUENCES for transcription factors
  10. Eukaryotic Promoter
    The two most commonly found consensus sequences are the GC box and the TATA box
  11. DO NOT Confuse DNA & Protein
    DNA sequences - 'sequence' 'promoter' 'box' 'element'

    • Proteins
    • 'factor' 'activator' 'repressor'
  12. Key Concept
    Promoter recognition in eukaryotes is carried out by accessory proteins, that in turn recruit RNA polymerase

    • General Transcription factors (GTFs)
    • ex: IID, IIA, IIB, etc.

    Together they comprise a 'basal txn apparatus'
  13. Formation of the Basal Apparatus
    1) Transcription factor IID = TFIID, binds the TATA box sequences

    TFIID is a holoenzyme with several subunits including TATA binding protein = TBP

    2) RNA poly II, other GTFs & a mediator complex bind to TFIID at the promoter

    3) Interactions b/w REGULATORY PROTEINS bound to upstream regulatory sequences and the BASAL APPARATUS influences LEVELS of txn.
  14. Key Concept
    The activity of the basal apparatus is influenced by upstream regulatory elements
  15. Key Concept
    The regulartory promoter of a gene can contain MULTIPLE ELEMENTS outside of the core promoter
  16. Key Concept
    Some regulatory elements functions at a DISTANCE from the core promoter, and can work in BOTH DIRECTIONS!

    enhancer elements
  17. Formation of the Basal Transcription Apparatus
    When all factors are present, the DNA is UNWOUND and RNA Pol starts making RNA from +1
  18. Elongation
    RNA Pol II and some associated factors MOVE DOWN the DNA making a pre-mRNA
  19. The Pre-mRNA is 'Capped'
    * A 7-methyl guanosine (7MG) is added. 5'-5' phosphate bond

    * Occues EARLY in elongation

    * Methyl groups (-CH3) are often added to the 2'-OHs of the next few nucleotides
  20. 5' Cap
    1) PROTECTS tge end of the mRNA from degradation

    2) Is required for TRANSLATION
  21. Termination
    1) RNA pol II transcripts are CUT downstream of a conserved sequence.

    2) A poly(A) polymerase adds a long run of AMPs onto the 3' end of the pre-mRNA
  22. THREE Modifications to Pre-mRNAs
    • 1) Addition of 5' cap (7MG)
    • - shortly after initiation
    • - stability
    • 2) Addition of 3' poly-A tail
    • - after termination
    • - stability
    • 3) Removal of intervening sequences
    • - RNA splicing
    • - after termination
  23. Key concept
    -Most eukaryotic genes containing BLOCKS of coding sequence (exons) and non-coding sequence (introns)

    -Both are present in the initial RNA transcript, but the introns are removed before export

    RNA splicing
  24. How were INTRONS discovered?
    Proof that introns exist came from DNA:RNA hybridization experiments
  25. HOW is RNA splicing accomplished?
    • - Must be PRECISE, to maintain the sequence of the polypeptide
    • - requires consensus sequences:
    • - at exon/intron JUNCTIONS
    • - WITHIN the intron
  26. Intron Consensus Sequence
    • - Short consensus sequences at each splice junctions
    • - A poorly conserved sequence WITHIN THE INTRON upstream (18-40 nts) of the 3' splice site
  27. Key Concept
    RNA splicing occurs through a 'lariat' intermediate
  28. The Splicesosome
    • - includes FIVE small nuclear ribonucleoprotein particles (snRNPs)
    • - they contain FIVE snRNAs (approx. 1-200 nts): U1, U2, U4, U5, U6
    • - snRNAs recognize mRNA splice sites by complementary base pairing!
    • - snRNAs also have catalytic activity
  29. Mechanisms of Splicing
    • 1) the 5' splice site is cut
    • - exon 1 is held in place by the spliceosome
    • - 5' end of intron attaches to branch point

    • 2) The 3' splice site is cut
    • - Exon 1 is joined to Exon 2 by a phosphodiester bond
    • - the lariat is released
  30. Key Concept
    Some genes are spliced in DIFFERENT ways, producing DIFFERENT mRNAs!

    Alternative Splicing

  31. Key Concept
    Alternate 3' cleavage can produce DIFFERENT RNAs from ONE gene.
  32. Non - mRNA May Be Spliced By Other Mechanisms
    - Some RNA molecules can self-splice (autocatacytic activity)

    • - Exs:
    • *Some ribosomal RNAs
    • * Some chloroplast and mitcochondrial RNAs
  33. Transfer RNA (tRNA)
    • - short chains (70-90 nucleotides long)
    • - have special secondary structure
    • - 'link' the mRNA to the polypeptide (crick's hypothesis)
    • - all tRNAs (proks/euks) are processed
    • - have more modified bases than any other class of RNA
    • - tRNA genes may be present in numerous copies
  34. Key Concept
    • Some RNA sequences are CHANGED after transcription!
    • - some bases are changed
    • - Some bases are CHANGED
    • - Some bases are ADDED or DELETED
    • - CONTRADICTS central dogma
    • RNA editing
  35. tRNAs are also Spliced
    • - by distinct splicing enzymes
    • - an intron in a pre- tRNA is CUT OUT, then the ends are LIGATED
    • - uses TWO enzymes
  36. So...
    All tRNAs are EXTENSIVELY PROCESSED to form the final functional molecules!
  37. Where do rRNAs come from?
    Genes can be present in MULTIPLE COPIES

    in euks, made in the nucleolus

    • Several different ones:
    • - proks: 3 rRNAs
    • - Euks: 4 rRNAs

    Also undergo PROCESSING

    Involves small nucleolar RNAs (snoRNAs)