LECTURE 14 RNA: TRANSCRIPTION

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LECTURE 14 RNA TRANSCRIPTION PROCESSING

• chapter 8
• announcements
• key ideas
• RNA
• transcription in Prokaryotes
• transcription in Eukaryotes

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ANNOUNCEMENTS

• missed or late for quiz ?
• same documentation required as for exams
• no makeup average of those you write
• exam 2 M 11.13 in class, as listed on calendar
• extra tutorial help
• M 300 430 F 1100 1230 in WHI 111
• extra-credit tutoring in progress, check web
  page
• extra-credit seminar WHI AUD F 330
• Dr. Juli Wade, Michigan State U.
• Hormonal and Genetic Influences on Sexual
  Differentiation of the Zebra Finch Song System

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CH8 KEY IDEAS

• 3 processes of information transfer in genetics

CH8 ?
CH9 ?
CH7 ?
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CH8 KEY IDEAS

• DNA transcription RNA translation
  protein

• translation requires transfer RNAs ribosomes
• information transfer by non-overlapping triplet
  code
• special DNA sequences signal initiation
  termination of transcription translation
• in Eukaryotes only...
• mRNA transcripts are processed prior to
  translation
• noncoding introns interrupt coding exon sequences
• introns are spliced out of 1 mRNA ? final mRNA

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CH8 KEY IDEAS

• transcription translation

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RNA

• DNA ? protein... RNA intermediate?
• pulse-chase experiments with RNA precursors...
  pulse radioactive, chase nonradioactive,
  autoradiograph

• ?synthesis of RNA in nucleus ? protein in
  cytoplasm

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RNA
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RNA
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RNA
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RNA
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RNA

• usually single-stranded, ? double-stranded,
  complex 3D structures antiparallel

RNA DNA

• ribose sugar ? deoxyribose

• uracil pyrimidine ? thymine

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RNA

• classes
• informational
• mRNA translated ? polypeptides
• functional
• tRNA bind transport amino acids
• rRNA components of ribosomes
• snRNA participate in modifying rRNA
• components of spliceosomes

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TRANSCRIPTION IN PROKARYOTES

• T2 bacteriophage infection of E. coli
• pulse-chase with radiolabeled uracil
  (RNA-precursor)
• labeled RNA recovered only immediately after
  pulse...
  ? rapid turnover
• phage-induced
  RNA similar to
  
  T2 DNA...
  ? DNA ? RNA

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TRANSCRIPTION IN PROKARYOTES

• in vitro synthesis shows RNA ? DNA... ? DNA
  template complementary RNA bases

• similar to DNA replication, with DNA
  polymerase... ? transcription enzyme... RNA
  polymerase ?

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TRANSCRIPTION

• RNA synthesized from 1 or both DNA strands?
• hybridization experiment
• complementary DNA strands have different
  densities
• each RNA hybridizes to only 1 DNA strand
• transcription is asymmetrical

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TRANSCRIPTION IN PROKARYOTES

• DNA template for mRNA transcription

• note directional nature of event

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TRANSCRIPTION IN PROKARYOTES

• DNA coding strand nontemplate strand
• sequence homologous to transcribed mRNA
• DNA has A, RNA has U
• DNA template strand ? mRNA

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TRANSCRIPTION IN PROKARYOTES

• genes transcribed from one but either DNA strand
• transcription by complementary pairing of bases
• catalyzed by RNA polymerase (RNA pol)

error!
error!
error!

• new mRNA grows 5' ? 3'
• RNA pol moves along DNA template strand 3' ? 5'

5' 3'
5' 3'
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TRANSCRIPTION IN PROKARYOTES

• normally illustrate in other direction for
  convenience

• new mRNA grows 5' ? 3'

• RNA pol moves along DNA template strand 3' ? 5'

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TRANSCRIPTION IN PROKARYOTES

• transcription of rRNA genes in Triturus nucleus

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TRANSCRIPTION IN PROKARYOTES

• 3 distinct stages of transcription
• 1. initiation
• 2. elongation
• 3. termination

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TRANSCRIPTION IN PROKARYOTES

• initiation
• 2 types of sequences in prokaryotic genes
• 1. promoter sequences signal initiation
• 2. coding sequences are transcribed

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TRANSCRIPTION IN PROKARYOTES

• initiation
• 2 regions of homology among promoter sequences
• consensus sequences RNA pol binding sites

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TRANSCRIPTION IN PROKARYOTES

• initiation
• ? factor binds to 10 and 35 consensus regions
• initiates melting or denaturing of DNA
• transcription begins when ? subunit dissociates

open promoter complex
closed promoter complex

• different ? factors recognize different DNA
  sequences

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TRANSCRIPTION IN PROKARYOTES

• elongation
• new mRNA grows 5' ? 3'
• RNA pol and transcription bubble moves 3' ? 5'
  along the DNA template strand

5' 3'
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TRANSCRIPTION

• elongation
• new mRNA grows 5' ? 3'
• RNA pol moves along DNA template strand 3' ? 5'
• ribonucleoside triphosphate added to 3' end of
  nth base

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TRANSCRIPTION IN PROKARYOTES

• elongation
• new mRNA grows 5' ? 3'
• RNA pol moves along DNA template strand 3' ? 5'
• pyrophosphate ion released

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TRANSCRIPTION IN PROKARYOTES

• termination
• RNA pol recognizes signal for chain termination
• 2 mechanisms for termination in prokaryotes
• 1. direct termination - termination sequence

mRNA 3' UTR DNA CG rich AAA...(6)
5' 3'
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TRANSCRIPTION IN PROKARYOTES

• termination
• direct termination, RNA pol recognizes 40 bp
  terminator sequence on template
• RNA forms hairpin loop
• poly-A tail bonds weak
• signal to release RNA pol
• ATP-independent

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TRANSCRIPTION IN PROKARYOTES

• termination
• RNA pol recognizes signal for chain termination
• 2 mechanisms for termination in prokaryotes
• 1. direct termination - termination sequence
• 2. rho-dependent

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TRANSCRIPTION IN PROKARYOTES

• termination
• rho-dependent termination
• no U-residues formed
• no hairpin loop
• rho binds to rut site
• on RNA

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TRANSCRIPTION IN PROKARYOTES

• termination
• rho-dependent termination
• no U-residues formed
• no hairpin loop
• rho binds to rut site
• on RNA
• rho pulls RNA from
• RNA pol

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TRANSCRIPTION IN PROKARYOTES

• termination
• rho-dependent termination
• no U-residues formed
• no hairpin loop
• rho binds to rut site
• on RNA
• rho pulls RNA from
• RNA pol

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TRANSCRIPTION IN EUKARYOTES

• differences because of complexity in Eukaryotes
• 1. RNA synthesis

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TRANSCRIPTION IN EUKARYOTES

• Prokaryotes
• RNA pol ? all types of RNA
• polycistronic mRNA
• Eukaryotes
• RNA pol I ? rRNA (except 5S rRNA)
• RNA pol II ? monocistronic mRNA, some snRNA
• RNA pol III ? tRNA, 5S rRNA, some snRNA

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TRANSCRIPTION IN EUKARYOTES

• Prokaryotes
• RNA pol only
• Eukaryotes
• RNA pol II general transcription factors
  (GTFs) ? mRNA

DNA GTFs
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TRANSCRIPTION IN EUKARYOTES

• differences because of complexity in Eukaryotes
• 1. RNA synthesis
• 2. RNA processing

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TRANSCRIPTION IN EUKARYOTES

• Prokaryotes
• no processing
• Eukaryotes
• processed before being transported to the
  cytoplasm

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TRANSCRIPTION IN EUKARYOTES

• differences because of complexity in Eukaryotes
• 1. RNA synthesis
• 2. RNA processing
• 3. chromosome organization
• 4. split genes

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TRANSCRIPTION IN EUKARYOTES

• Prokaryotes
• naked DNA (nearly)
• Eukaryotes
• chromatin euchromatin heterochromatin

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TRANSCRIPTION IN EUKARYOTES

• 3 distinct stages of transcription
• 1. initiation
• 2. elongation
• 3. termination

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TRANSCRIPTION IN EUKARYOTES

• initiation
• GTFs ? TATA sequence before RNA pol II binding
• attract RNA pol II
• positions complex
• GTFs added after ? preinitiation complex
• transcription bubble

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TRANSCRIPTION IN EUKARYOTES

• elongation
• GTFs added after ? preinitiation complex
• transcription bubble
• RNA pol II carboxyl tail domain (CTD)
  phosphorylated...

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TRANSCRIPTION IN EUKARYOTES

• 2 types of processing
• cotranscriptional

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TRANSCRIPTION IN EUKARYOTES

• cotranscriptional processing CTD dependend
• guanyltransferase adds 7'-methylguanosine cap
  to 5' end of mRNA
• protects single stranded RNA from degradation

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TRANSCRIPTION IN EUKARYOTES

• cotranscriptional processing CTD dependend
• splicing by spliceosomes (... stay tuned)

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TRANSCRIPTION IN EUKARYOTES

• termination
• AAUAAA sequence near 3' end initiates cleavage

• ... by endonuclease 20 bp downstream

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TRANSCRIPTION IN EUKARYOTES

• 2 types of processing
• cotranscriptional
• posttranscriptional

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TRANSCRIPTION IN EUKARYOTES

• posttranscriptional processing
• poly(A) polymerase adds poly(A) tail of 150-200
  adenosine residues to 3' end cleavage site

• complete 1 mRNA

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EUKARYOTIC RNA

• 1 mRNA shortened before transport to cytoplasm
• chicken ovalbumin DNA/mRNA hybrid ?
• coding sequences... exons

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EUKARYOTIC RNA

• 1 mRNA shortened before transport to cytoplasm
• chicken ovalbumin DNA/mRNA hybrid ?
• coding sequences... exons
• intervening sequences... introns

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EUKARYOTIC RNA

• 1 mRNA shortened before transport to cytoplasm
• chicken ovalbumin DNA/mRNA hybrid ?
• coding sequences... exons
• intervening sequences... introns
• introns spliced from mRNA
• genomic DNA ? RNA exons introns

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TRANSCRIPTION IN EUKARYOTES

• summary...
• genomic DNA ? RNA exons introns
• transcribed ? 1 mRNA transcript
• processed ? cap polyadenylation
• spliced ? splicing intermediate
• spliced ? mature mRNA

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TRANSCRIPTION IN EUKARYOTES

• 1 gene ? multiple functions by alternative
  splicing
• multiple gene functions in different...
• tissues
• developmental stages
• different mRNAs from same 1 mRNA transcript...
  e.g., ?-tropomyocin gene...

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TRANSCRIPTION IN EUKARYOTES

• 1 gene ? multiple functions by alternative
  splicing e.g., ?-tropomyocin gene...

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TRANSCRIPTION IN EUKARYOTES

• gene splicing mechanism...
• sequence homologies at exon-intron splice
  junctions
• consensus sequences for splicing enzymes...

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TRANSCRIPTION IN EUKARYOTES

• spliceosome
• snRNA aligns sequences correctly for splicing

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TRANSCRIPTION IN EUKARYOTES

• splicing reaction
• intron lariat structure
• introns excised by 2 transesterification reactions

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TRANSCRIPTION IN EUKARYOTES

• splicing reaction
• intron lariat structure
• introns excised by 2 transesterification
  reactions
• snRNA proteins ? small ribonuclear particles
  (snRNPs)
• snRNP 1 mRNA transcript ? spliceosome
• spliceosome catalyzes splicing transesterification
  reactions

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TRANSCRIPTION IN EUKARYOTES

• self splicing mechanisms, mRNA catalyzes reaction
  (called ribozymes)

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TRANSCRIPTION IN EUKARYOTES

• self splicing mechanisms, mRNA catalyzes reaction
  
• spliceosome-catalyzed mechanism

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QUESTIONS CHAPTER 8

• TUTORIAL 6