Transcription and Translation

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Transcription and Translation

• Chapter 16

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Information Flow in the Cell
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Transcription

• The stages of transcription are
• Initiation
• Elongation
• Termination

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Transcription in Bacteria
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Transcription

• First step in converting genetic information into
  proteins.
• Copying DNA to make an RNA messenger
• DNA-directed synthesis of RNA
• Catalyzed by RNA polymerase
• Follows the same base-pairing rules as DNA,
  except that in RNA, uracil substitutes for thymine

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Transcription
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RNA Polymerase

• Pries the DNA strands apart and hooks together
  the RNA nucleotides
• Synthesizes the RNA strand in the 5' ? 3'
  direction
• RNA strand is complementary to the DNA template
  strand.
• Provides single-stranded RNA copy of the DNA
• This strand is the template strand, the other the
  non-template strand.

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Bacterial RNA Polymerase

• Globular enzyme with several channels and a
  magnesium atom in its active site
• Holoenzyme made up of the core enzyme, which has
  the ability to synthesize RNA
• And a regulatory sigma protein, which is required
  for initiation of transcription
• Which is a co-enzyme

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Bacterial RNA Polymerase
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Sigma Factors

• Binds to the core enzyme, enabling RNA polymerase
  to recognize and bind to specific sites on DNA,
  called promoters.
• Sigma is the factor responsible for identifying
  the start site of a gene
• Different sigma factors are activated in response
  to different environmental conditions

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Promoters

• Two key regions
• The 10 box has the sequence 5'-TATAAT-3
• located 10 bases upstream of the transcription
  start site
• The 35 box has the sequence 5'-TTGACA-3
• located 35 bases upstream of the transcription
  start site
• Sigma identifies the 10 and 35 promoter sites,
  properly orienting the RNA polymerase core
  complex for transcription at the gene start site

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How Transcription Begins
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How Transcription Begins
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How Transcription Begins
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Elongation and Termination

• Sigma dissociates from the core enzyme once the
  initiation phase of transcription is completed
• RNA polymerase moves along the DNA template in
  the 3' ? 5' direction
• Synthesizes RNA in the 5' ? 3' direction
• RNA polymerase encounters a transcription
  termination signal
• Causes the RNA to form a hairpin structure

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Transcription in Eukaryotes
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Eukaryotic Polymerases

• Contains many polymerases depending on gene type

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Initiation of Translation

• Promoter- specific sequence of DNA where
  polymerase attaches and transcription begins
• Upstream of the gene
• Promoters signal the initiation of RNA synthesis
• Transcription factors
• Help eukaryotic RNA polymerase recognize promoter
  sequences

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Initiation of Transcription

1. Promoter (TATA box)
2. Several transcription factors bind to DNA before
   polymerase
3. Polymerase binds to transcription factors
4. Transcription starts

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Elongation

• As RNA polymerase moves along the DNA
• It continues to untwist the double helix,
  exposing about 10 to 20 DNA bases at a time for
  pairing with RNA nucleotides
• Enzyme adds at 3 end
• Many polymerases can work on the same strand to
  produce many copies

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Termination

• Differs in prokaryotes and eukaryotes
• Prokaryotes- terminator sequence causes
  polymerase to detach and mRNA to detach
• Eukaryotes- polymerases keep on adding
  nucleotides but proteins associated with the mRNA
  cut it free from the growing strand

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Alteration of mRNA

• Cap and Tail
• The 5? end receives a modified nucleotide cap
• For recognition
• The 3? end gets a poly-A tail
• Protects RNA from Degradation

A modified guanine nucleotide added to the 5? end
50 to 250 adenine nucleotides added to the 3? end
Polyadenylation signal
Protein-coding segment
5?
3?
AAAAAA
G
P
P
AAUAAA
P
Stop codon
Start codon
Poly-A tail
5? Cap
3? UTR
5? UTR
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Alteration of mRNA

• RNA splicing- Removes introns and joins exons

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Introns

• The presence off introns
• Allows for alternative RNA splicing
• Regulatory function
• May be the reason humans have so few genes
• Proteins often have a modular architecture
  consisting of discrete structural and functional
  regions called domains
• Different exons can code for the different
  domains in a protein

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Introns
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Eukaryotic vs. Prokaryotic Transcription
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Translation
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Translation

• RNA-directed synthesis of a polypeptide
• A cell translates an mRNA message into protein
  with the help of transfer RNA (tRNA)

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Bacterial Translation

• Ribosomes catalyze translation of the mRNA
  sequence into protein.
• In bacteria, ribosomes begin translation of an
  mRNA before RNA polymerase has finished
  transcribing it

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Eukaryotic Translation

• In eukaryotes, mRNAs are synthesized and
  processed in the nucleus and transported to the
  cytoplasm for translation by ribosomes

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Role of Transfer RNA

• Each tRNA carries a specific amino acid that is
  transferred to protein
• The addition of amino acids to tRNAs is mediated
  by aminoacyl tRNA synthetase
• Molecules of tRNA are not all identical
• Each carries a specific amino acid on one end and
  an anticodon on the other end
• Consists of a single RNA strand that is only
  about 80 nucleotides long
• Is roughly L-shaped

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tRNA
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Ribosomes

• Ribosomes can be separated into two subunits, the
  large subunit and the small subunit
• tRNAs are found on three sites in ribosomes
• A site is the acceptor site for the
  aminoacyl-tRNA
• P site holds the growing polypeptide chain
• E site is where tRNAs no longer bound to an amino
  acid exit the ribosome

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Ribosomes

1. A peptide bond forms between the amino acid on
   the aminoacyl-tRNA in the A site and the existing
   polypeptide held on the tRNA in the P site
2. Polypeptide is transferred to the tRNA in the A
   site.
3. The ribosome moves forward to the next codon
4. tRNA in the E site exits the ribosome

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Ribosomes

1. The tRNA in the P site moves into the E site
2. The tRNA with the polypeptide chain moves from
   the A site to the P sit
3. A new aminoacyl-tRNA enters the A site.

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Ribosomes
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Initiation of Translation

• Translation begins at the AUG start codon
• Complementary to a section of one rRNA in the
  small ribosomal subunit
• Once the small ribosomal subunit is bound to the
  mRNA, the aminoacyl initiator tRNA binds to the
  AUG sequence
• The large subunit binds and completes the
  initiation complex. The initiator tRNA is located
  in the P site of the ribosome

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Initiation
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Elongation

• At the start of elongation, the initiator tRNA is
  in the P site, and the E and A sites are empty
• An aminoacyl tRNA binds to the codon in the A
  site via complementary base pairing between
  anticodon and codon, and peptide bond formation
  occurs
• Etc, etc
• The ribosome translocates down the mRNA by three
  nucleotides, and the tRNA attached to the growing
  protein moves into the P site

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Polyribosomes

• Strings of ribosomes, assemble along an mRNA to
  increase the rate of protein production

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Elongation

• The three steps in elongation
• arrival of aminoacyl tRNA
• peptide bond formation
• translocationrepeat down the length of the mRNA.

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Termination

• When the A site encounters a stop codon, a
  release factor enters the site and catalyzes
  hydrolysis of the bond linking the tRNA in the P
  site with the polypeptide chain

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Termination
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Post-Translational Modifications

• Most proteins go through an extensive series of
  processing steps before they are ready to go to
  work in a cell.
• Get into proper conformation
• Molecular chaperones speed folding of the protein
• Small chemical groups may be added to eukaryotic
  proteins in the rough endoplasmic reticulum

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Post-Translational Modifications

• Some proteins receive a sorting signal to ensure
  that the molecule will be carried to the correct
  location in the cell
• Some proteins are augmented with sugar or lipid
  groups that are critical for normal functioning.
• Many proteins are altered by enzymes that add or
  remove a phosphate group
• Switch the protein from an inactive state to an
  active state or vice versa

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The Molecular Basisof Mutation
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Mutation

• Any change in an organisms DNA sequence. DNA
  mutations affect phenotype only when the mutation
  is expressed
• Resulting protein functions abnormally
• Not all mutations affect the proteins ability to
  function and thus do not generate a phenotype

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Point Mutation

• Change in a single nucleotide
• Most common
• Can result from errors in DNA replication or from
  exposure to mutagenic toxins

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Missense Mutation

• A point mutation that causes a change in the
  amino acid sequence of the protein
• Sickle-cell anemia results from a missense
  mutation in the hemoglobin gene

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Other Mutations
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Other Mutations