Transcription

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Transcription

• Chapter 26

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Genes

• Nucleotide seqs w/in DNA
• 2000 genes for peptides in prokaryotes
• 50,000 genes for peptides in eukaryotes
• DNA not DIRECT template for peptides
• DNA template for RNA (mRNA)
• Synth mRNA from DNA transcription
• So DNA transcribed to mRNA
• mRNA used to translate genetic code ? peptide
  (next lecture)

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RNA Similar to DNA

• Both nucleic acids
• Both composed of 4 nucleotides A, G, C
• BUT RNA has U, not T
• Both have ribose sugar
• BUT RNA - ribose, DNA - deoxyribose
• Both linked by phosphodiester bonds
• ? Sugar-phosphate backbone
• BUT RNA in single-strand (not dbl helix)
• Strand can fold back on itself
• Can form intrastrand helices, other 2o structures

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Transcription DNA ? RNA Similar to
Repln DNA?DNA

• Complementarity
• Base seq daughter DNA complementary to DNA
  template (parent) strand
• Base seq mRNA complementary to DNA template
  strand
• Initiation, Elongation, Termination Processes

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• Polymerases catalyze syntheses new nucleic acids
• Free 3 OH attacks PO4 of incoming triphosphate
• Pyrophosphate (PPi) reld
• (NMP)n NTP ? (NMP)n1 PPi

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• Template strand read 3 ? 5
• So copied strand synthd 5 ? 3
• Complementary strands antiparallel
• DNA double helix must be unwound in both
• Topoisomerases impt to relieve tension on helix

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Transcription DNA?RNA Different than
Repln DNA?DNA

• Amt DNA copied
• Repln entire chromosome
• Both strands dbl helix
• Transcrn only 1 gene (part of chromosome) from
  1 strand of double helix
• ? Single strand mRNA
• BUT gene copied more than once
• Yields many transcripts of same gene

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• Each DNA strand transcrd has diff name
• RNA transcribed from DNA template strand
• Complementary strand of dbl helix called DNA
  nontemplate strand or coding strand
• Same seq as RNA transcript, except for one
  difference
• How is it different from transcript??

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• Both strands can serve as template
• Each codes for own proteins

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• Origin
• Repln one origin in E. coli
• Whats that called?
• Transcrn Enzs/prots must know where along
  length of DNA to begin copying/stop copying
• Polymerase
• Repln DNA polymerase
• Several types w/ varied subunits
• Has proofreading ability
• Requires primer
• Elongation up to 1,000 nucleotides/sec

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• Polymerase contd
• Transcrn RNA polymerase
• 1 complex w/ 6 subunits
• Holoenzyme
• 1 subunit (s) directs rest of enz to site of
  initiation of transcrn

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• Transcrn RNA polymerase contd
• No proofreading
• No primer needed
• Begins mRNA w/ GTP or ATP
• Elongation 50-90 nucleotides/sec
• Unwinding
• Repln helicases used
• Transcrn RNA polymerase keeps 17 bps unwound

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E. coli Promoter Region

• DNA seq _at_ which transcrn apparatus comes
  together to begin copying the gene
• So each gene has promoter
• Extends from 70 to 30 w/ respect to RNA start
  for many genes
• Consensus DNA seqs -- highly conserved in both
  seq and location

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• Consensus DNA seqs contd
• 1 base first nucleotide to be transcribed
• Usually purine
• What are the purines??
• -10 region (toward 3 end of template strand) 6
  nucleotide seq w/ consensus TATAAT
• Spacer 16-18 nucleotides
• -35 region 6 nucleotide seq w/ consensus TTGACA
• -40 ? -60 region AT-rich region Up-stream
  Promoter (UP element)

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• When pattern met exactly
• RNA polymerase recognizes most efficiently
• Get rapid transcription
• When pattern varies from consensus
• Longer for RNA polymerase to recognize promoter
• So longer time of transcription

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Initiation E. coli Transcrn

• s subunit of RNA polymerase searches for promoter
  region
• Scans 2000 nucleotides/ 3 sec along template
  strand
• Holoenzyme binds at promoter region ? closed
  complex
• DNA bound to holoenzyme is intact

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• About 15 bps unwound _at_ -10 region ? open
  complex
• Probably conforml changes in polymerase enz
  assist in opening

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• Now transcrn initiated w/ nucleotides paired to
  template strand, added to polymer
• After 8-9 nucleotides added, s subunit dissocs
• Can scan another region to find another promoter

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Regulation of Transcrn

• Strength of consensus at promoter region, as
  mentioned
• Some polymerases have gt1 s subunit
• Cell stress ? use of diff s subunit, specific for
  partic promoters needed to alleviate specific
  stresses
• Ex heat shock genes

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• Proteins may bind DNA seqs in/around promoter
• Some attract RNA polymerase to promoter region
• Activate transcrn of these genes
• Some block RNA polymerase from binding _at_ promoter
• Repressors -- repress transcrn of these genes

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• Proteins respond to metab, repro, stress
  conditions w/in cell
• Conds may require much peptide or depletion of
  peptide
• REMEMBER Mechs by cell to regulate
  glycolysis/metab??

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Elongation of Transcrn in
E. coli

• Holoenzyme freely moves along template chain
• Freer w/ dissocn of s subunit
• Forms transcription bubble
• Contains holoenzyme, template strand, new RNA
  strand

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• New RNA strand transiently base-paired to
  template DNA strand ? DNA-RNA hybrid

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• DNA helix rewinds behind transcription bubble

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• Error rate in transcrn 1/104-105 bases added
• Much higher than in repln
• Acceptable
• Cell will make many transcripts of same gene
• Most ? proper (active) peptides
• Some ? improper peptides can be accommodated by
  cell
• What if template strand were mutated?

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Termination Transcrn in E. coli

• Need RNA polymerase to be processive
• If falls off, must re-start _at_ promoter
• What might happen in cell if problem w/ RNA
  polymerase processivity?
• BUT may pause _at_ certain template strand seqs
• Some template strand seqs cause RNA polymerase
  to stop

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• Two termination types in E. coli
• Rho (r) independent
• Template seq ? RNA transcript w/
  self-complementary nucleotides
• 15-20 nucleotides
• G-C rich, followed by A-T rich regions
• Transcript forms stable hairpin loop
• Template has string of A nucleotides ? string of
  U nucleotides in transcript _at_ 3 end
• Causes RNA polymerase to pause may just bypass
  site and continue transcripn

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• OR transcript changes conformn, hairpin
  stabilizes ? disruption hybrid
• ? RNA transcript dissociates
• Polymerase dissocs
• DNA helix reanneals, rewinds

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• r dependent
• r protein termination factor
• Binds RNA transcript _at_ partic binding sites
• Moves along new transcript 5 ? 3 to transcrn
  bubble
• RNA-DNA helicase activity promotes translocation
• Has ATP hydrolysis ability
• Finds elongation paused
• Disrupts DNA-RNA hybrid
• Mech unkown

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Prokaryote Transcription

• Prokaryote chromosome in cytoplasm
• No organized nucleus
• Prokaryote chromosome simple
• mRNA transcrd directly from DNA seq
• No introns/exons junk DNA etc.
• As mRNA synthd, almost immediately translated ?
  peptide

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EukaryoteTranscription

• More complex, less understood
• 3 RNA polymerases I, II, III
• Each w/ specific function
• Each binds diff promoter seq
• RNA Polymerase I
• Transcribes some rRNAs
• Pre-ribosomal RNA precursors for 18S, 5.8S, 28S
  rRNAs

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• RNA Polymerase III
• Transcribes tRNAs and 5S rRNA
• Some promoters found w/in gene itself

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• RNA Polymerase II
• Transcribes mRNA (most impt to transcrn process)
• Many subunits sim to E. coli
• Largest subunit has long carboxyl-terminal tail
• Consensus aa seq
• Carboxyl-terminal domain (CTD)
• Sepd from main enz body by linker

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• Recognizes many promoters
• TATA box (TATAAA) in euks
• Inr seq
• Requires transcrn factors

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Transcription Factors

• Impt for RNA polymerase II
• Proteins
• Modulate binding of RNA polymerase II to promoter
  region
• Complex w/ RNA polymerase ? proper binding to
  template, proper elongation

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Transcrn Occurs in the Nucleus

• mRNA transcript ? cytoplasm for transln
• For peptides used outside nucleus
• REMEMBER nuclear membr has pores
• Euk genes complicated
• REMEMBER introns/exons, junk? DNA
• Polymerase doesnt seem to distinguish
• Euk DNA transcrd directly ? mRNA
• ? 1o transcript directly reflecting entire gene
  and any introns/junk

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• mRNA must be processed
• From 1o transcript to mature mRNA that
  successfully codes for protein
• Also impt for bactl tRNAs
• Some impt processing steps catalyzed by RNAs (not
  enzymes)
• Ribozymes

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• For functioning peptide, intron seqs excised
  before translation
• 1o mRNA transcripts spliced, rejoined
• Transesterification reaction
• Sim to topoisomerase mech

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• Intron seqs excised
• Most nuclear mRNAs spliced by specialized
  RNA-protein complexes
• snRNPs small nuclear RiboNucleoProteins
• About 5 RNAs (U1-U5, each 100-200 bps) 50
  prots complex ? spliceosome

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• Addl snRNPs join ? spliceosome
• Pulls U1, U2 close, so exon ends close

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• Sets up attack by exon OH end ? phosphate _at_
  other exon end

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• Get lariat structure of intron seq nucleotides

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• Lariat cleaved, released w/ snNRPs
• Phospho-diester backbone of mRNA reannealed

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• Processing occurs during transcrn
• CTDcarboxyl terminal domain of RNA polymerase
• CBCcap-binding complex

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5 Cap

• 7-Methylguanosine added
• Unusual 5, 5-triphosphate linkage
• Sev enzs impt (Cap synthesizing complex)
• Assocd w/ CDT of RNA polymerase II
• Cap Binding Complex holds cap to CDT
• May be impt in initiation of translation
• Protects mRNA from ribonucleases

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3 polyA Tail

• 80-250 adenylate residues
• Binding site for partic prots
• May stabilize mRNA against enz destruction
• Also found on prok mRNAs
• Conserved cleavage-site seqs signal placement of
  enz complex for polyA tail synth

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• Catd by enzyme complex
• Endonuclease cleaves some mRNA ? free 3 hydroxyl
• Polyadenylate polymerase adds nucleotides
• Other prots also impt

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• Final transcript mature mRNA

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Alternative Processing ? Diff Proteins from Same
Gene

• Diff mRNAs may result from varied processing of
  same gene
• Only some genes
• Molec signals in 1o transcript identify these
  mRNAs
• Give rise to diff prots

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• Vary by
• gt1 cleavage/polyA site
• Impt for diversity in variable region of Ig heavy
  chains
• Alternative splicing patterns
• Found in diff myosin heavy chains
• Both
• Found in two related prots in rats
• Need RNA binding prots

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Inhibition of Transcription by
Antibiotics

• Actinomycin D
• Planar, non-polar
• Intercalates between nucleotide bases of DNA
• Esp. between G-Cs in G-C rich seqs
• Now polymerase cant move along DNA template

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