gene expression

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gene expressionfrom DNA to protein

• biology 1

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• Genes control metabolism
• Gene expression is a two stage process
• transcription
• translation
• Genes consists of triplets of nucleotides - the
  genetic code
• Protein synthesis in prokaryotes and eukaryotes
• Eukaryotic modification of RNA
• Mutations

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Genes control metabolism

• One gene-One polypeptide rule
• Polypeptides that are constructed as a result of
  transcription/translation process become either
• structural proteins
• enzymes
• Those proteins that have quaternary structure may
  have polypeptides originating from different genes

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The transcription/translation process

• Transcription DNA codes for the construction of
  mRNA
• Translation mRNA is read by rRNA at a ribosome
  tRNA brings amino acids to ribosome as defined by
  code on mRNA
• Ribosome assembles polypeptide
• Recap on RNA - a ribose nucleic acid that uses
  Uracil (U) in place of Thymine (T)

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The genetic code

• The linear sequence of nucleotides in DNA
  ultimately determines the linear sequence of
  amino acids in a polypeptide
• There are approximately 20 types of amino acid to
  choose from
• In DNA, the four nucleotides are ATCG
• Therefore, the sequence of four possible
  nucleotides must code for 20 amino acids
• If DNA used a individual nucleotide to refer to
  an individual amino acid, this system would only
  code for 41 amino acids
• Using two nucleotides would account for 42 16
  amino acids
• Using three nucleotides would account for 43 64
  amino acids
• Since there are only 20 amino acids, yet 64
  possible codes, some redundancy occurs

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• Each block of three nucleotides, ultimately
  corresponding to a particular amino acid, is
  called a codon
• In the first stage of the gene expression
  process, transcription, the information in the
  codons of a gene are transferred to mRNA
• This process is via an RNA polymerase that uses
  one of the DNA strands of the double helix (the
  template strand)
• For each amino acid, there are generally several
  codons possible. Also, some codons have a
  non-amino acid equivalent, but instead send
  specific messages to RNA polymerase (start/stop)

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Transcription

• Three phases
• Polymerase binding and initiation
• Elongation
• Termination
• In eukaryotes, RNA polymerase II bind to specific
  regions on DNA called promoters
• Promoters are typically 100 nucleotides long,
  including
• The initiation site, where transcription begins
• Nucleotides sequences that help initiate
  transcription

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• Initiation in eukaryotes requires transcription
  factors, DNA-binding proteins that bind to
  specific nucleotide sequences in the promoter
  region
• A common place for a transcription factor to bind
  is the TATA box
• RNA polymerase recognizes the promoter site once
  DNA and transcription factor have bound at the
  TATA box
• RNA polymerase temporarily separates the double
  helix for transcription

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• In elongation, RNA polymerase II (eukaryotes)
• Untwists the DNA molecule
• Adds incoming RNA free-floating nucleotides to
  the 3 end of the RNA strand (grows 5 to 3)
• mRNA grows at 30-60 nucleotides/sec. The mRNA
  chain starts to peel away as the double helix
  reforms
• Followed in series, several molecules of RNA
  polymerase can simultaneously transcribe the same
  gene
• Transcription proceeds until the polymerase
  reaches a termination code

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Translation

• During translation, proteins are synthesized
  according to a genetic message of sequential
  codons along mRNA
• tRNA (transfer RNA) interprets between the base
  sequence in mRNA and the amino acid sequence in a
  polypeptide chain. To do this
• Transfer amino acids from cytoplasm to ribosome
• Recognize the correct codons on mRNA
• Molecules of tRNA are specific to one particular
  amino acid
• One end of tRNA attaches to a specific amino acid
  (3 end)
• The other end attaches to an mRNA codon by base
  pairing with its anti-codon

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• An anti-codon is a nucleotide triplet in tRNA
• tRNA decodes the genetic message codon by codon
• There are 45 types of tRNA, which is sufficient
  for the 64 codes, since there is a relaxation of
  base-pairing on the third nucleotide (wobble)
• e.g., U in 3rd position of anticodon can bind
  with A or G on the equivalent codon
• In some cases, third position on a tRNA anticodon
  is occupied by Inosine (a sixth nucleotide) that
  can bind with U, C or A

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• Joining of tRNA to specific amino acid at the 3
  end is by Aminoacyl-tRNA synthetase
• Each amino acid has a particular synthetase
  enzyme
• ATP activates the amino acid by losing 2
  phosphate groups, and joining to the amino acid
  as AMP
• tRNA bonds to the amino acid, which loses AMP
• Ribosomes coordinate the pairing of tRNA
  anticodons to mRNA codons
• Consist of 2 subunits (small and large) that
  remain separated when not involved in protein
  synthesis
• Ribosomes are composed of 60 rRNA and 40 protein

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• In addition to an mRNA binding site, two further
  sites on a ribosome are the P- and A-sites
• P-site holds the tRNA carrying the growing
  polypeptide chain
• A-site holds the tRNA that has the next amino
  acid in the polypeptide sequence
• Building of a polypeptide chain consists of three
  steps
• Initiation
• Elongation
• Termination

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Translation Initiation

• In eukaryotes, the small ribosomal unit binds to
  an initiator tRNA (methionine anticodon UAC)
• The small ribosomal unit binds to the 5 end of
  mRNA, and in doing so brings the tRNA anticodon
  in close proximity with mRNA methionine codon
• This binding requires initiation factors
• Finally, the large subunit binds to the complex
• The initiator tRNA fits to the p-site of the
  ribosome
• The vacant a-site is ready for the next
  aminoacyl-tRNA complex

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Translation elongation

• Codon recognitionmRNA codon in the a-site of the
  ribosome forms hydrogen bonds with anti-codon of
  an entering tRNA carrying the next amino acid in
  the chain
• Peptide bond formationThe enzyme peptidyl
  transferase (part of the large ribosomal unit)
  catalyzes the peptide bond between the incoming
  amino acid and the growing polypeptide chain
• Translocationthe tRNA in the p-site releases
  from the ribosome, and the tRNA in the a-site
  moves into the vacated site

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Translation termination

• A termination codon signals the end of
  translation by binding to a protein release
  factor, this causes
• Peptidyl transferase hydrolyzes the bond between
  the completed polypeptide and the tRNA in the
  p-site
• This frees the polypeptide and tRNA so that they
  can release from the ribosome
• The two ribosomal units disassociate
• mRNA may continue to be translated by
  polyribosomes

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Differences between prokaryotic and eukaryotic
gene expression

• Lack of nuclear membrane in prokaryotes means
  that transcription can occur at one end of the
  mRNA molecule, while translation can be occurring
  at the other end
• In eukaryotes, RNA is modified following
  transcription before translation
• 5 cap added (modified guanine nucleotide
• Poly-A tail added (200 adenine nucleotides) to 3
  end
• These ends might protect mRNA sequence (attaching
  to untranslated leader and trailer sequences
  respectively)
• Gene splicing

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Gene splicing

• Eukaryotic mRNA has segments of non-code, called
  introns (code sequences called exons)
• Introns and exons are initially coded into one
  long strand called hnRNA (heterogenous RNA)
• In RNA splicing, introns are removed from hnRNA
  to make mRNA
• Process of splicing mRNA involves SnRNPs
  (snurps) - small nuclear ribonucleoproteins,
  that are composed of SnRNA (small nuclear RNA)
  and proteins
• Together with extra proteins, SnRNPs form
  complexes called spliceosomes, which excise
  introns (SnRNPs attach to either end of each
  intron)
• tRNA and rRNA also need to be spliced, but
  different agents do the splicing - ribozymes, RNA
  molecules that act as enzymes (note thus not all
  enzymes are proteins)

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• Why do introns exist?
• May regulate gene activity
• Splicing may regulate export of mRNA to cytoplasm
• Introns cause exons to be further apart, and
  therefore to be further away from each other on
  the chromosome this could mean a higher
  probability of recombination during cross-over
• Specific introns may code for specific domains
  within a protein

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When things go wrong...
MUTATION!

• Mutation a permanent change in DNA that can
  involve large chromosomal regions or a single
  nucleotide pair
• Point mutation a mutation limited to one or two
  nucleotides in a single gene
• Base-pair substitution
• Missense mutation
• Nonsense mutation
• Insertion/deletion mutations

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• Base-pair substitutions generally have no effect
  if they occur on the third nucleotide of a
  triplet
• If they do change the amino acid, one a.a.
  substitution may not radically affect the
  functionality of the final polypeptide
• In some cases, functionality is improved in most
  cases, functionality is impaired
• In nonsense mutations, the substitutions causes a
  triplet to read STOP, abruptly terminating
  polypeptide chain. Such mutations are usually
  harmful

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• Insertions or deletions add or remove one or more
  nucleotides from a sequence
• Since a reading frame for nucleotides is based on
  a series of three, insertions and deletions that
  add or remove a sequence of nucleotides not
  divisible by 3 can substantially alter the final
  polypeptide
• Such a mutation is referred to as a frameshift -
  these mutations usually result in non-functional
  proteins, unless they occur towards the end of a
  sequence