From Gene to Protein Chapter 17

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From Gene to ProteinChapter 17

• Associate Professor Pamela L. Pannozzo
• Principles of Biology I BSC 1010

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How does DNA determine an organismss traits?

• DNA codes for proteins and RNA
• One gene one polypeptide
• Proteins determine phenotypes via chemical
  reactions in cells
• Gene expression the process by which DNA
  directs protein synthesis. Two stages
• Transcription
• Translation
• All cells do not express the same genes

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Stages of Gene Expression

• Transcription
• DNA information transcribed into sequence of mRNA
  nucleotides--A, G, C, U
• Occurs in the nucleus
• Produces single-stranded mRNA
• Translation
• mRNA information translated into sequence of
  amino acids
• Occurs in the cytoplasm on ribosomes (free or
  bound)
• Produces polypeptide
• If not translated?RNA results

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Transcription Initiation

• RNA Polymerase II binds at promoter site,
  beginning transcription unit
• Opens DNA 10-20 bases at a time, moves along
  pairing complementary nucleotides
• Free nucleotides from nucleoplasm

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Transcription Initiation (cont)

• TATA sequence commonly 25 nucleotides from
  transcription start point
• Several transcription factors bind to promoter
  before RNA Polymerase II in forming transcription
  initiation complex
• Once RNA Pol II is firmly attached, unwinds
  transcription bubble

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Transcription Elongation

• RNA Pol II moves downstream elongating the mRNA
  transcript
• RNA Pol II works in 5 (-PO4) to 3 (-OH)
  direction
• 60 nucleotides per second added

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Transcription Termination

• mRNA transcript disengages at 10-35 nucleotides
  past polyadenylation signal (AAUAA) (pre-mRNA)
• RNA Polymerase II falls off of DNA
• Pre-mRNA transcript processed to mature mRNA
• Several RNA Polymerase II transcribe a single
  gene at the same time

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Transcription

• Original DNA code TACCGCTTCAGAATT
• What will be the mRNA code?

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DNA TACCGCTTCAGAATT mRNA AUGGCGAAGUCUUAA
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Pre-mRNA Processing

• Ends altered (protection, ribosome binding)
• To both sides of UTR--untranslated region
• 5 capmodified Guanine
• 3 end poly-A tail50-250 adenines
• Middle parts removed
• Non-coding introns cut out
• mRNA spliced back together

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Why Remove the Middle Parts?

• Eukaryotic genes fragmented
• Exons--sequence coding for amino acids
• Introns--non-coding sequences
• Average DNA transcription unit 8,000 nucleotides
• Average mature mRNA transcript 1,200 nucleotides

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• Different combinations of exons can generate
  different polypeptides via alternative splicing

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RNA Splicing

• Splicing signal is short nucleotide sequence at
  ends of introns
• Recognized by snRNPS in nucleus (RNA proteins),
  form spliceosomes
• Intron may catalyze its own excision

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Translation

• Converts order of mRNA nucleotides to order of
  amino acids
• Genetic Code governs rules of translation
• Every three nucleotide sequence (codon) codes for
  an amino acid
• Ribosomes read mRNA by codons

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Genetic Code is Universal

• Living things share the same nucleotides
• And produce the same amino acids
• Enables transplantation of DNA between organisms
• Proteins from one species can be produced by
  another species!
• Example-tobacco plant expresses firefly gene

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Ribosomes

• Two subunits
• rRNA and proteins

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tRNA
Hydrogen bonding causes hairpin loops

• Bring amino acids to the ribosome
• Two ends
• Anticodon
• 3 OH end attached amino acids
• Specific amino acid joined to tRNA by
  aminoacyl-tRNA synthetase
• Amino acid corresponds to mRNA codon

3-D shape
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Translation Initiation

• mRNA binds to small ribosomal subunit and
  tRNA-met
• Ribosome scans for AUG start codon
• Large subunit joins (initiation complex aided by
  initiation factor proteins)
• mRNA threads through 5 end first via elongation
  factors

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Translation Elongation

• Elongation factors bring tRNA with anticodon
  matching codon
• tRNA enters A site -gt P site -gt E site
• Amino acids added one by one to growing
  polypeptide chain

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Fig. 9.15 How translation works
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Translation Termination

• Process continues until a stop codon (UAA, UAG,
  UGA) enters the A site
• Release factor facilitates release of polypeptide
• Ribosome complex falls apart and the polypeptide
  is released

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Fig. 9.18 How protein synthesis works in
eukaryotes
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Translation (cont)

• GTP hydrolysis provides energy
• Polyribosome strings enhance polypeptide
  production

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Free or Bound Ribosome?

• Freecytosolic proteins
• Boundendomembrane system and secreted proteins
• Translation begins on free ribosomes?polypeptides
  with signal peptide cause ribosome to be
  transported to ER membrane, become bound

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Post-translation modifications of polypeptide

• Folding (chaperone proteins assist)
• Chemical modification of some amino acids
• Cleavage of polypeptide
• Joining of polypeptides as protein subunits
• Occurs in cytoplasm

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Versatility of RNA

• Machinery of protein synthesis is RNA
• More versatile than DNA
• Functions
• Catalyze intron excision (ribozymes)
• Process pre-mRNA (small nucleolar RNA)
• Control gene expression (small interfering- and
  microRNA)
• Transcribe DNA (mRNA)
• Pair codons and amino acids (tRNA)
• Translate mRNA (rRNA)

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Mutations

• Changes in the genetic material of a cell
• Chromosomal rearrangements (change in positioning
  of genetic material)
• Point mutations (change in content of genetic
  material)
• Substitutions
• Cause missense or nonsense
• Insertions and Deletions
• Cause frame-shift
• Both provide the raw material for evolution

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Mutations (cont)

• Mutations in germ line cells can be inherited
• Mutations in somatic cells are not inherited, but
  passed to descendant cells
• Causes
• Errors in DNA Replication (1/1010 nucleotides)
• Mutagens
• Short-wave (high energy) electromagnetic
  radiation
• Mutagenic chemicals

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Substitution
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