From Gene to Protein

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From Gene to Protein

• CHAPTER 17

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DNA ? RNA ? protein

• transcription synthesis of messenger RNA (mRNA)
  from a DNA template
• occurs in nucleus
• translation synthesis of a polypeptide from the
  mRNA template created by transcription
• occurs at ribosomes in cytoplasm

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The Genetic Code

• flow of info from gene to protein is based on a
  triplet code (3-nucleotide long words called
  codons)
• near universality
• evolutionary significance

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Transcription

• Initiation
• begins at a promoter sequence on the DNA template
  strand
• Elongation
• RNA polymerase unwinds DNA links RNA
  nucleotides as they base-pair with the DNA
  template strand
• Termination
• occurs at a terminator sequence in prokaryotes

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Initiation

• promoter sequence
• commonly include a TATA box in eukaryotes
• serves as binding site for RNA polymerase
  mediated by transcription factors in eukaryotes
• determines which DNA strand serves as the
  template

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Elongation

• RNA polymerase can only add nucleotides to the 3'
  end of a growing RNA molecule (this does not
  require a primer as in DNA replication)
• base-pairing rules

DNA A C T G RNA U G A C
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Termination

• prokaryotes
• terminator sequence on DNA template strand
  signals RNA polymerase to detach
• mRNA is available for immediate use (no
  modification necessary)
• eukaryotes
• proteins cut the transcript free about 10-35
  nucleotides downstream from a polyadenylation
  signal (AAUAAA)
• the resulting pre-mRNA is modified before
  leaving the nucleus

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pre-mRNA processing

• alteration of the pre-mRNA ends
• 5' end a modified guanine nucleotide is added
  (5' cap)
• 3' end 50-250 adenine nucleotides are added
  (poly-A tail)
• modifications of the ends help the mRNA leave the
  nucleus, protect it from degradation by
  hydrolytic enzymes, help ribosomes attach to
  the 5' end

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pre-mRNA processing cont.

• RNA-splicing removal of introns (non-coding
  regions) joining of exons (coding regions)
• carried out by spliceosomes which are made up of
  snRNPs (small nuclear RNA proteins) associated
  with additional proteins
• alternative RNA-splicing
• increases number of proteins
• an organism can make

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Translation

• basic mechanism
• as mRNA is moved through a ribosome, codons are
  translated into amino acids
• this is accomplished by base-pairing tRNA
  molecules with complimentary anticodons to the
  mRNA codons deliver the appropriate amino acids
  which are joined together by the ribosome
• 3 stages
• initiation, elongation, termination

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transfer RNA (tRNA)

• Structure

• 20 different aminoacyl-tRNA synthetases join the
  20 different amino acids to the correct tRNAs
• there are about 45 different tRNAs
• wobble base-pairing rules are relaxed
  (usually at 3rd position) allowing some tRNAs to
  bind to more than one codon

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Ribosomes

• composed of 2 subunits made in the nucleolus
• constructed of proteins and ribosomal RNA (rRNA)
• subunits join only when attached to mRNA
• 3 binding sites for tRNA
• E site discharge used tRNAs
• P site holds tRNA carrying growing polypeptide
  chain
• A site holds tRNA carrying the next amino acid
  to be added to the chain
• catalyzes the formation of peptide bonds between
  amino acids
• polypeptide released thru an exit tunnel

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Initiation

• requires proteins called initiation factors
• involves joining of mRNA, first tRNA, the
  ribosome subunits
• small ribosomal subunit binds to mRNA initiator
  tRNA (carrying Met) and scans the mRNA until it
  reaches the start codon (AUG), then the large
  ribosomal subunit joins

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Elongation

• amino acids are added one by one to the growing
  polypeptide chain
• requires proteins called elongation factors
• 3-step cycle
• codon recognition tRNA anticodon pairs with
  mRNA codon in A site
• peptide bond formation the polypeptide chain on
  the tRNA in the P site is bound to the amino acid
  on the tRNA in the A site
• translocation tRNA in P site is moved to E site
  released tRNA in A site is moved to P site A
  site is now open for the next tRNA

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codon recognition
translocation
peptide bond formation
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Termination

• when a stop codon (UAG, UAA, UGA) reaches the A
  site, a release factor binds to it causing the
  addition of a water molecule to the polypeptide
  chain which hydrolyzes the chain allowing it to
  be released from the ribosome thru the exit tunnel

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Polyribosomes

• a single mRNA can make many polypeptides
  simultaneously because several ribosomes can
  translate it at the same time

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Post Translation

• polypeptide chains fold into their functional
  tertiary structure
• tertiary proteins may join with other tertiary
  proteins becoming the subunits of a protein with
  quaternary structure
• some amino acids may be chemically modified
• enzymes may remove amino acids from the leading
  end or cleave a polypeptide chain into two pieces

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Rough ER Ribosomes

• translation always begins on ribosomes in the
  cytoplasm
• if the mRNA codes for a protein destined for cell
  secretion or the endomembrane system, the
  ribosome will attach to the rough ER
• the cue to do so comes from a signal peptide (20
  amino acid sequence) in the growing polypeptide
  chain that is recognized by signal-recognition
  particle (SRP) in the cytoplasm

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Mutations changes in DNA

• point mutations changes in a single base-pair
• substitution replacement of one nucleotide
  its partner with another pair of nucleotides
• can be silent have no effect on the encoded
  protein
• are usually missense change a single amino acid
  in the encoded protein
• can be nonsense change a codon to a stop codon
  causing translation to terminate early (shortens
  encoded protein)
• insertion addition of nucleotide pairs
• deletion loss of nucleotide pairs
• when insertions deletions are not a multiple of
  3, they significantly alter the reading frame
  (change all codons downstream of the mutation)
  and are called frameshift mutations causing
  extensive missense

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Mutagens

• physical chemical agents that react with DNA in
  ways that cause mutations
• examples X-rays, UV rays, carcinogens