Transcription

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Chapter 12

• Part 2

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Transcription

• Proteins are end product of many genes, while
  determining the genetic code, other tried to
  figure out how DNA nucleic acid can specify
  protein made of AA
• Synthesize RNA from DNA template
• mRNA complementary to the gene sequence of either
  of the 2 DNA strands mRNA then codes for AA
• Transcription is initial step in the process of
  information flow

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Observations for RNA as Intermediate

• DNA is in the nucleus but protein is made in the
  cytoplasm by the ribosome
• no direct interaction with protein synthesis
• RNA made in the nucleus and is chemically similar
  to DNA
• RNA migrates to cytoplasm site of protein
  synthesis
• Amount of RNA is generally proportional to amount
  of protein in cell

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• Information stored in DNA is transferred to RNA
  intermediate that then moves on to participate in
  protein synthesis
• studies done in bacteria and phages

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RNA Polymerase

• Found in rat liver in 1959 and had the same
  general substrate requirements as DNA polymerase
• use ribose instead of deoxyribose and no need for
  a primer
• Start with NTP and stays that way and another
  added to this
• Linked 5 to 3 during phosphodiester bond
  formation

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RNA Polymerse (continued)

• RNA pol in E coli has an ?, ? and ? and ?
  factor makes a holoenzyme
• ? and ? provide catalytic basis and active site
  for transcription
• ? factor plays a regulatory function in
  initiation of RNA transcription, only one form of
  enzyme but may be several ? factor
• Eukaryotic RNA polymerase 3 distinct forms,
  more complex than bacteria

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Promoters, Template Binding and the ? Factor

• Transcribe strand of DNA, template strand and
  complement strand is the partner strand
• Template binding is the 1st step
• binding occurs when the ? factor recognizes
  specific DNA sequence called promoters 5
  region upstream of the gene
• once recognize the gene, the DNA is denatured and
  DNA is accessible for transcription

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Promoters Govern Efficiency of Initiation

• Strong and weak promoters variation in time of
  initiation
• mutations can severely reduce initiation of gene
  expression
• 3 important points involved in promoter-enzyme
  interactions
• consensus sequences of DNA 2 sequences in
  bacterial promoter
• TATAAT 10 bases upstream of gene (-10) or
  Pribnow box
• TTGACA 35 bases upstream of gene
• cis-acting elements next to or on the same side
  
• trans-acting elements molecules that bind these
  elements
• degree of RNA pol binding to different promoters
  leads to variable gene expression, sequence
  variation in the promoter
• ? factor major form is ?70 recognized by most
  promoter there are also alternative forms, each
  recognize different prompters, specificity of
  initiation of transcription

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Initiation and Elongation

• Once RNA pol binds promoter, begins initiation
  adding 1st 5-ribonucleotide that is
  complementary to the DNA nucleotide
• Continue to add ribonucleotides in the 5 to 3
  manner reading the DNA in the 3 to 5 direction-
  elongation
• Make a RNA/DNA hybrid that is antiparallel

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Dissociation of ? Factor

• After a few ribonucleotides are added, ? factor
  dissociates from holoenzyme (50 nt/seq _at_37?)
• Eventually get the entire gene transcribed until
  it reaches the termination sequence, close to the
  start of the next gene

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Termination

• May be dependent on termination factor ? (rho)
• ? interacts with the transcript at a specific
  sequence that triggers the release of the
  transcript and enzyme from the DNA
• RNA pol will dissociate into its subunits until
  the next gene
• Bacterial genes are clustered all but the last
  gene lack the termination signal, make a large
  mRNA that encodes more than one protein
• polycistronic mRNA genes transcribe in this
  manner are usually all needed for the same
  process
• eukaryotes are monocistronic one message and
  one protein

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Eukaryotic Differences in Transcription

• Transcription is in the nucleus and under the
  influence of 3 RNA pol, doesnt interact with
  ribosome until completed and moves out of nucleus
• Requires the uncoiling of compact chromatin fiber
  to access the gene by RNA pol and other
  regulatory proteins
• chromatin remodeling

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• 3. More extensive interaction between
  cis-acting upstream sequences and trans-acting
  protein factors involved in stimulating and
  initiating transcription
• enhancers (promoter and other control units) in
  the 5 regulatory region upstream, within gene or
  in the 3 downstream beyond the coding region
• Eukaryotic mRNA must be modified from a 1
  transcript to mRNA
• processing

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Processing

• Must add a 5 cap and a 3 tail
• Other modifications occur to the internal
  nucleotide sequences
• 1 transcript or pre-mRNA is much longer than the
  mRNA that is eventually translated
• found in the nucleus heterogeneous nuclear RNA
  (hnRNA) and complexed with proteins forming
  heterogeneous nuclear ribonucleoprotein particles
  (hnRNPs)
• only about 25 turns into mRNA
• remove excess sequence and splice back together
  exported and translated split genes and splicing

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3 RNA Polymerases

• RNA pol make unique RNAs in various areas of the
  nucleus
• Structure is more complex than prokaryotes 2
  large subunits and 10-15 smaller subunits

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RNA pol II

• Transcribes all the mRNA in the nucleus
• Interacts with several cis-acting elements to aid
  efficient transcription
• Goldberg-Hogness (TATA) box - 35 bp upstream
• heptapeptide of A and T (similar to the -10 TATA
  in prokaryotes) may be responsible for fixing
  site of initiation
• CAAT box - 80 bp upstream consensus sequence
  GGCCAATCT, may also be other upstream regulatory
  sequences may also be present
• DNA regions called enhancers another cis-acting
  element
• upstream of promoter, downstream of gene or
  within gene
• modulate transcription from a distance
• may not bind template to enhance transcription

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Trans-Acting Factors

• Facilitate template binding to initiate
  transcription called transcription factors
• TFIIA, TFIIB, ..
• RNA pol II needs these because they cant bind
  promoter sites
• TFIID binds directly at TATA box and often called
  the TATA binding protein (TBP) has 10 peptide
  subunits
• once binds DNA there is a sequential binding of
  at least 7 other transcription factors to TFIID
  pre-initiation complex that then binds RNA pol II
• transcription factors supplant the ? factor in
  bacteria

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Caps and Tails

• hnRNA has similarities to mRNA in the cytoplasm
• Transcription makes a large RNA that is processed
  before moving to cytoplasm

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Post-Transcriptional Modification

• Addition of the cap 7-methylguanosine (7mG) cap
  is added to the 5 end almost immediately after
  this is made
• may function to protect the transcript and to
  help transport the mRNA to the cytoplasm
• complex process probably why protective as
  there isnt an enzyme that recognizes this type
  of bond
• Addition of CH3 to the ribose of the 1st 2
  nucleotides of RNA
• hnRNA and mRNA may have 250 adenylic acid
  residues added at the 3 end (tail) called a
  polyA tail
• add the polyA tail 10-35 nt downstream of AAUAAA
  sequence, if mutated the tail isnt added and RNA
  is rapidly degraded
• found on the end of almost all mRNA with the
  exception of the histone genes

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Additional Modifications

• Once the cap and tail are added can complete
  process and move to cytoplasm
• Coding regions are interrupted by intervening
  sequences called introns
• find these in the genes but they are not
  translated in the protein
• genes with introns are called split genes
• RNA fragments kept to make mRNA are exons
• must excise intron and fuse exons together

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Identification of Introns

• Used hybridization of mRNA to DNA
• introns looped out 7 in chicken ovalbumin gene
• Direct comparison of nucleotide sequences of DNA
  with mRNA to find the precise sequence of
  intervening sequences
• most genes have introns

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(No Transcript)
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Splicing Mechanisms

• Different mechanisms for different types of RNA
  and the RNA in mitochondria and chloroplasts
• Group I introns rRNA
• need not additional components transcript is
  responsible for removal ribozyme
• use G as a co-factor to cut the intron out and
  refuse the ends to make rRNA
• Group II introns used in mRNA and tRNA in
  mito/chloro similar to group I but doesnt use G

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Splicosome

• Nuclear derived mRNA requires a more complex
  system
• Sequence at the ends of the intron are similar
  5-GU and 3-AG attract a complex called the
  splicosome
• Contains a unique set of small nuclear RNAs
  (snRNAs) that complex with proteins to make
  snRNPs or snurps
• found only in the nucleus and are rich in Us U1
  bonds and then recruits others splicing
  commences
• the 3-OH on A within the branch point region of
  intron interacts with U2 and forms a lariat
  form the intron takes
• ligate exons and snRNPs release

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Alternative Splicing

• Splicing can be a possible regulatory step in
  gene expression
• depending on the introns removed to yield
  different mRNA
• Alternative splicing results in a series of
  related proteins called isoforms

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RNA Editing of the Final Transcript

• Another form of post-translational modification
  RNA editing
• Sequence of pre-mRNA is actually changed prior to
  translation
• substitution editing identities of individual
  nucleotides are altered in nuclear RNAs and
  prevalent in mito/chloro RNA in plants
• insertion/deletion editing nucleotides are
  added ot subtracted from total number of bases
  used in mtDNA of Trypanosoma African sleeping
  sickness

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Multiple Copies of mRNA in Bacteria

• See many RNA molecules being made as well as
  ribosomes starting attach and make proteins