Bmsc 220 6b Transcription and RNA processing

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  1. to transcribe, RNApolymerase II requires  that a gene contains:
    • downstream
    • -exons (coding);
    • -introns (noncoding);
    • -a stop sequence

    • upstream
    •  -core promoter region (Inr and TATA or TATA-like box;)
  2. Transcription factors
    proteins that help RNA polymerase II transcribe genes
  3. general transcription factors
    • -core promoter and INR
    • -essential for RNA pol II binding
  4. gene-specific transcription factors
    • -not part of the general machinery 
    • -bind to promoter and enhancer elements
    • -control the rate of transcription

    • ex:
    • -upstream promoters
    • -enhancers
  5. TATA-binding proteins
    general transcription factors that bind to TATA sequence before and after RNA pol II does

    • before: TFIID TFIIF
    • after: TFIIE and TFIIH
  6. What must happen before RNA pol II can bind to the TATA sequence?
    what happens before transcription startes
    -general transcription factors called TATA-binding proteins (TBPs TFIID andT FIIF) must bind to the TATA box.

    -After RNA pol binds, two more TBPs bind (TFIIE and TFIIH), and transcription can start
  7. Gene-specific promoter and enhancers
    • -proteins like transcription factors
    • -bind to promoter and enhancer sequences on the DNA to alter the rate of transcription (by affecting the efficiency of the pre-initiation complex).
  8. Enhancer sequences
    -either downstream or upstream of transcription start site

    -gene specific 

    -bind transcription factors which ↑ or ↓ the amount of the transcript (mRNA)that is made

    – modulate the rate of transcriptional regulation by regulating the pre-initiation complex
  9. •Mutations in regulatory DNA sequences or transcription factors leads to:
    • human disease
    • sequences: beta thalassema

    factors: Rett syndrome
  10. Transcription factors:
    structural properties:
    • -have a modular structure
    • -Can function as transcriptional activators and transcriptional repressors

    -contain separate DNA-binding and activation/repression domains

    -contain other domains that regulate their activity or allow them to interact with other transcription factors
  11. Mediator protein complex
    • - large protein complex
    • -plays a key role in linking the general transcription factors to the gene-specific transcription factors
    • – binds to the pre-initiation complex
  12. DNA looping
    • -DNA folds on itself
    • -allows transcription factors bound to a distant enhancer to interact with proteins in the RNA polymerase/ Mediator complex at the core promoter
  13. Release of RNA polymerase from the basal (pre initiation) complex  to initiate transcription requires:
    the phosphorylation and helicase activities of TFIIH:

    -Phosphorylation of  C-terminal domain (CTD) on RNA pol II

    -Helicase must break hydrogen bonds in dsDNA for RNA pol II to start working
  14. transcriptional activators:
    2 general mechanisms of action
    • 1.  Interact with mediators and general transcription factors 
    • 2.  Interact with co-activators, which modify chromatin structure.
  15. transcriptional repressors
    -Blocking binding of transcriptional activator to DNA sequence element

    -Repressing mediator activity

    -Interaction with co-repressors, which serve to modify chromatin structure into a non-permissive state
  16. •Actively transcribed chromatin exists in a relatively ___ state
  17. Histone code:
    • a) Histone tail acetylation and deacetylation  
    • b) Other histone modifications
  18. Histone acetylase (HAT)
    • histone tail acetylation:
    • -addition of acetyl groups to specific lysine residues in histone tails.
    • -transcriptionally permissive chromatin
  19. Histone deacetylase (HDAC)
    • histone tail deacetylation (HDAC):
    • non-permissive state
  20. HATs
    • -co-activators
    • -must bind to activator first
  21. HDACs
    • Co-repressors
    • -must bind to either a repressor first
  22. Chromatin remodeling factors
    -increase availability of a gene to be transcribed by displacing the nucleosome
  23. post-transcriptional processing
    • •processing of the pre-mRNA to the mature RNA
    • -occurs in the nucleus
    • -allows for extra level of regulatory control
  24. Processing of Eukaryotic messenger RNAs
    • transcription
    • 5' capping
    • 3' polyadenylation
    • splicing
  25. •Pre-mRNA
    the primary transcript that is processed to form messenger RNA in eukaryotic cells.
  26. 5' capping
    -A 7-methylguanosine cap is first added during the modification of the 5' end of a transcript, often before transcription has ceased.

    -increases stability (“half-life”) of mRNA (makes the 5’ end look like a 3’ end so them RNA will not be degraded by 5’ exonucleases)
  27. 3' polyadenylation
    -addition of a Poly-A tail to 3' end of eukaryotic mRNAs by the enzyme poly-A polymerase.

    -protectsthe mRNA molecule from enzymatic degradation in the cytoplasm, aids intranscription termination, helps the export of mRNA from the nucleus
  28. poly-A tail
    a tract of about 200 adenine nucleotides
  29. What directs where Poly A will occur?
    A DNA sequence along with a cleavage by endo nuclease
  30. Small nuclear RNAs (snRNAs)
    • -nuclear RNAs that range in size from 50 to 200 bases and form the RNA component of the spliceosome.
    • -assist in targeting snRNPS to splice junctions through hybridization to RNA transcript 
    • -are catalytic and carry out steps in the splicing reaction

    • ex:
    • U1, U2,U4, U5, and U6 snRNAs
  31. •Small nuclear ribonucleoprotein particles (snRNPs)
    - complexes of snRNAs with proteins that play central roles in the splicing process.

    • Ex:
    • U1, U2, U3, U5, and U4/U6 snRNPs
  32. Spliceosomes
    • -are large complexes composed of proteins and RNAs (snRNAs)
    • -carry out the splicing reactions
  33. SR slicing factors
    • -bind to exon sequences to direct where a splicesome will form
    • -will differ in two different cell types for which a specific mRNA is alternatively spliced
    • -assist in determining the final “choice” of exons that will appear in any one cell type for any one alternatively spliced mRNA
  34. Alternative splicing
    • -generation of different mRNAs by varying the pattern of pre-mRNA splicing
    • -allows many different combintions
    • -allowed by presence of introns between exons in a gene

    • ex
    • •In the sex determination mechanisms of Drosophila, alternative splicing of the same pre-mRNA determines whether a fly is male or female.
  35. Dscam gene
    -expressed as a cell surface adhesion molecule(marker) important in allowing neurons to find their target cells

    -contains four sets of alternative exons, with a single exon from each set being incorporated into the spliced mRNA.
  36. 5.8S, 18S, and 28S rRNA: (RNA pol I)
    • -makes up the majority of cellular RNA (over 80% of total cellular RNA)
    • -are the products of one unique gene (45S rRNA gene) that is present in multiple repeated copies within the genome
  37. •pre-rRNA
    the primary transcript that is cleaved to form individual ribosomal RNAs—the 28S, 18S, and 5.8S rRNAs of higher eukaryotic cells.
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Bmsc 220 6b Transcription and RNA processing
Bmsc 220 6b Transcription and RNA processing
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