Protein Synthesis

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Protein Synthesis
AKA Gene Expression
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Overview

• Protein Synthesis/Gene Expression
• Transcription
• Initiation
• Elongation
• Termination
• Translation
• Initiation
• Elongation
• Termination
• Mutations
• Causes and rate
• Mutagens
• Physical
• Chemical
• Viral
• Point mutation
• Insertions and deletions
• Substitution

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Protein Synthesis

• DNA holds information in the form of a nucleotide
  sequence.
• The DNA inherited by an organism makes the
  organism unique because it dictates the synthesis
  of unique set of proteins
• Gene expression the synthesis of a unique set
  of proteins
• The process of gene expression includes two
  stages, called transcription and translation

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Transcription and Translation

• Central dogma of molecular biology
• DNA ??RNA ??protein
• Transcription
• Is the production of a specific (m)RNA molecule
  off of a DNA template
• The sequence of nucleotides in the mRNA
  corresponds to the sequence of nucleotides in the
  piece of DNA
• Takes place in the nucleus

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Transcription and Translation

• Translation
• Is the synthesis of a polypeptide (protein) using
  an mRNA template
• The sequence of amino acids in the polypeptide
  corresponds to the sequence of nucleotides in the
  mRNA
• Occurs on ribosomes in the cytoplasm

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Transcription and Translation

• In a eukaryotic cell the nuclear membrane
  separates transcription from translation
• RNA processing has to occur in the nucleus before
  the mRNA molecule can go to the cytoplasm

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RNA vs. DNA

• RNA is single stranded, not double stranded like
  DNA
• RNA is short, only 1 gene long, where DNA is very
  long and contains many genes
• RNA uses the sugar ribose instead of deoxyribose
  in DNA
• RNA uses the base uracil (U) instead of thymine
  (T) in DNA.

-Venn diagram -Protein Synthesis map
Table 17.1
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Transcription

• Transcription is the DNA-directed synthesis of
  RNA
• RNA synthesis
• Is catalyzed by RNA polymerase, which pries the
  DNA strands apart and hooks together the RNA
  nucleotides
• Follows the same base-pairing rules as DNA,
  except that in RNA, uracil substitutes for
  thymine

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Synthesis of an RNA Transcript

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

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Synthesis of an RNA Transcript - Initiation

• Promoter a region/sequence on the DNA strand
  that marks where transcription should begin
• Transcription factors bind to the promoter and
  help RNA polymerase to recognize where to begin
  transcription

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Synthesis of an RNA Transcript - Elongation

• RNA polymerase synthesizes a single strand of RNA
  against the DNA template strand

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Synthesis of an RNA Transcript - Termination

• A termination sequence in the DNA signals that
  transcription should end
• When one of these is encountered by the
  polymerase, the RNA transcript is released from
  the DNA and the double helix can zip up again.

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Transcription Overview
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• http//www.youtube.com/watch?vh3b9ArupXZg

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Alteration of mRNA Ends

• RNA processing occurs in the nucleus. After
  this, the messenger RNA moves to the cytoplasm
  for translation.
• Changes made to the mRNA strand include 1.
  Addition of a protective cap to the 5 end2.
  Addition of a poly-A tail to the 3 end 1 and
  2 protect the mRNA from enzymes in the
  cytoplasm that would degrade it3. Splicing
  removal of introns

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RNA Processing - Splicing

• The original transcript from the DNA is called
  pre-mRNA.
• It contains transcripts of both introns and
  exons.
• Introns non-coding, nonsense regions used for
  regulatory purposes
• Exons regions that code for genes
• The introns are removed by a process called
  splicing to produce mature messenger RNA (mRNA)

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RNA Processing

• In summary, a pre-mRNA is converted into a mature
  mRNA by doing 2 things
• Add protective bases to the ends
• Cut out the introns

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Translation

• Translation is the RNA-directed synthesis of a
  polypeptide
• Translation requires
• mRNA
• Ribosomes - Ribosomal RNA
• Transfer RNA
• Codons

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The Genetic Code

• Genetic information is encoded as a sequence of
  nonoverlapping base triplets, or codons

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The Genetic Code

• Codon A sequence of three nucleotides which
  together form a unit of genetic code in a DNA or
  RNA molecule.
• 3 nucleotides 1 codon 1 amino acid
• Since there are 4 bases (ACGU) and 3 positions in
  each codon, there are 4 x 4 x 4 64 possible
  codons
• There are 64 codons but only 20 amino acids.
• Therefore, most amino acids can be coded for by
  more than 1 codon
• 3 of the 64 codons are used as STOP signals they
  are found at the end of every gene and mark the
  end of the protein
• One codon is used as a START signal it is at the
  start of every protein
• Universal in all living organisms

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The Genetic Code

• A codon in messenger RNA is either translated
  into an amino acid or serves as a translational
  start/stop signal

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Transfer RNA

• Consists of a single RNA strand that carries a
  specific amino acid on one end and has an
  anticodon on the other end
• A special group of enzymes pairs up the proper
  tRNA molecules with their corresponding amino
  acids.
• tRNA brings the amino acids to the ribosomes.

The anticodon is the 3 RNA bases that matches
the 3 bases of the codon on the mRNA molecule
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Transfer RNA

• 3 dimensional tRNA molecule is roughly L
  shaped

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Ribosomes

• Ribosomes facilitate the specific coupling of
  tRNA anticodons with mRNA codons during protein
  synthesis
• Ribosomes are made of 2 ribosomal subunits which
  are constructed of proteins and rRNA

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Building a Polypeptide
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Building a Polypeptide

• We can divide translation into three stages
• Initiation
• Elongation
• Termination
• In General
• The AUG start codon is recognized by the
  methionyl-tRNA or Met
• Once the start codon has been identified, the RNA
  is decoded by tRNA molecules, which adds amino
  acids into a polypeptide chain
• The ribosome Translation ends when a stop codon
  (UAA, UAG, UGA) is reached

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Initiation of Translation

• The initiation stage of translation brings
  together 1. mRNA 2. tRNA bearing the first
  amino acid of the polypeptide3. two subunits of
  a ribosome

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Elongation of the Polypeptide Chain

• In the elongation stage, amino acids are added
  one by one to the preceding amino acid

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Termination of Translation

• The final stage is termination when the ribosome
  reaches a stop codon in the mRNA

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Translation

• The final step in translation is termination.
  When the ribosome reaches a STOP codon, there is
  no corresponding transfer RNA.
• Instead, a small protein called a release
  factor attaches to the stop codon.
• The release factor causes the whole complex to
  fall apart messenger RNA, the two ribosome
  subunits, the new polypeptide all disperse.
• The messenger RNA can be translated many times,
  to produce many protein copies.

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A summary of transcription and translation in a
eukaryotic cell
Figure 17.26
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Post-translation

• The new polypeptide is now floating loose in the
  cytoplasm if translated by a free ribosme.
• It might also be inserted into a membrane, if
  translated by a ribosome bound to the endoplasmic
  reticulum.
• Polypeptides fold spontaneously into their active
  configuration, and they spontaneously join with
  other polypeptides to form the final proteins.
• Sometimes other molecules are also attached to
  the polypeptides sugars, lipids, phosphates,
  etc. All of these have special purposes for
  protein function.

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Protein Synthesis Quiz

• 1. How does DNA hold information?
• 2. What is a promoter?
• 3. Where does translation take place?
• 4. What three things take happen to the pre-mRNA
  during RNA processing?
• 5. What do release factors do?
• 6. What enzyme catalyzes transcription?
• 7. What is a termination sequence?
• 8. AUG is the _________________.

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Protein Synthesis Quiz

• 1. In its specific sequences of nucleotides
• 2. a sequence on the DNA strand that marks where
  transcription should begin
• 3. At a ribosome in the cytoplasm
• 4. addition of a cap, addition of a poly-A tail,
  splicing (or removal of the introns)
• 5. Cause the ribosome and new protein to break
  off of the mRNA strand
• 6. RNA polymerase
• 7. A sequence on the DNA that marks where
  transcription should end
• 8. Start Codon

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Mutations Lab Quiz

• What is CF and what does it affect?
• Describe a mutation from your lab that would have
  led to CF.
• Describe a mutation from your lab that would not
  have lead to CF.
• Which race is most likely to develop CF? Which
  race is least likely to develop CF?

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Mutations Lab Quiz

• What is CF?
• Cystic fibrosis is a disease passed down through
  families that causes thick, sticky mucus to build
  up in the lungs, digestive tract, and other areas
  of the body.
• Describe a mutation that would have led to CF.
• A mutation in which the DNA sequence was changed
  but the corresponding amino acid was not.
• Describe a mutation that would not have lead to
  CF.
• A mutation in which the DNA sequence was changed
  and so was the corresponding amino acid sequence
• Which race is most likely to get CF? Least
  likely?
• Caucasians are most likely, Asians are least
  likely

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Lab Review

• Cystic fibrosis (CF) is a genetic disorder that
  particularly affects the lungs and digestive
  system
• Causes mucus in the lungs to build up
• Normally, mucus in the lungs traps germs, which
  are then cleared out of the lungs. But in CF, the
  thick, sticky mucus and the germs it has trapped
  remain in the lungs, which become infected.

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Lab Review

• The inherited CF gene directs the body's
  epithelial cells to produce a defective form of a
  protein called CFTR (or cystic fibrosis
  transmembrane conductance regulator) found in
  cells that line the lungs, digestive tract, sweat
  glands, and genitourinary system.

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Lab Review

• When the CFTR protein is defective, epithelial
  cells can't regulate the way chloride (part of
  the salt called sodium chloride) passes across
  cell membranes. This disrupts the essential
  balance of salt and water needed to maintain a
  normal thin coating of fluid and mucus inside the
  lungs, pancreas, and passageways in other organs.
  
• The mucus becomes thick, sticky, and hard to move.

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Lab Review

• Of all ethnic groups, Caucasians have the highest
  inherited risk for CF, and Asian Americans have
  the lowest.
• In the United States today, about 1 of every
  3,600 Caucasian children is born with CF.
• 1 of every 17,000 African Americans and only 1 of
  every 90,000 Asian Americans.

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Lab Review

• Of all ethnic groups, Caucasians have the highest
  inherited risk for CF, and Asian Americans have
  the lowest.
• In the United States today, about 1 of every
  3,600 Caucasian children is born with CF.
• 1 of every 17,000 African Americans and only 1 of
  every 90,000 Asian Americans.

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Lab Review

• Treatment includes prescription of digestive
  enzymes, antibiotics, and mucus thinning drugs
• A new treatment for CF, which is still being
  researched, is an inhaled spray containing normal
  copies of the CF gene. These normal genes deliver
  the correct copy of the CF gene into the lungs of
  CF patients.

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Mutation Causes and Rate

• The natural replication of DNA produces
  occasional errors. DNA polymerase has an editing
  mechanism that decreases the rate, but it still
  exists.
• Typically genes incur base substitutions about
  once in every 10,000 to 1,000,000 base pairs.
• Since we have about 6 billion bases of DNA in
  each cell, virtually every cell in your body
  contains several mutations.
• However, most mutations are neutral have no
  effect.
• Only mutations in cells that become sperm or
  eggsare passed on to future generations.
• Mutations in other body cells only cause trouble
  when they cause cancer or related diseases.

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• Earless Bunny
• http//www.huffingtonpost.com/2011/06/09/earless-r
  abbit-japan_n_873552.html?viewprintcomm_reffals
  e

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Point mutations

• Point mutations involve alterations in the
  structure or location of a single gene.
  Generally, only one or a few base pairs are
  involved.
• Point mutations can signficantly affect protein
  structure and function
• Point mutations may be caused by physical damage
  to the DNA from radiation or chemicals, or may
  occur spontaneously
• Point mutations are often caused by mutagens

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Mutagens

• Mutagens are chemical or physical agents that
  interact with DNA to cause mutations.
• Physical agents include high-energy radiation
  like X-rays and ultraviolet light

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Mutagens

• Mutagens are chemical or physical agents that
  interact with DNA to cause mutations.
• Physical agents include high-energy radiation
  like X-rays and ultraviolet light
• Chemical mutagens fall into several categories.
• Chemicals that are base analogues that may be
  substituted into DNA, but they pair incorrectly
  during DNA replication.
• Interference with DNA replication by inserting
  into DNA and distorting the double helix.
• Chemical changes in bases that change their
  pairing properties.

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Mutagens

• Mutagens are chemical or physical agents that
  interact with DNA to cause mutations.
• Physical agents include high-energy radiation
  like X-rays and ultraviolet light
• Chemical mutagens fall into several categories.
• Chemicals that are base analogues that may be
  substituted into DNA, but they pair incorrectly
  during DNA replication.
• Interference with DNA replication by inserting
  into DNA and distorting the double helix.
• Chemical changes in bases that change their
  pairing properties.
• Tests are often used as a preliminary screen of
  chemicals to identify those that may cause cancer
• Most carcinogens are mutagenic and most mutagens
  are carcinogenic.

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Viral Mutagens

• Scientists have recognized a number of tumor
  viruses that cause cancer in various animals,
  including humans
• About 15 of human cancers are caused by viral
  infections that disrupt normal control of cell
  division
• All tumor viruses transform cells into cancer
  cells through the integration of viral nucleic
  acid into host cell DNA.

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Point Mutation

• The change of a single nucleotide in the DNAs
  template strand leads to the production of an
  abnormal protein

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Types of Point Mutations

• Point mutations within a gene can be divided into
  two general categories
• Base-pair substitutions
• Base-pair insertions or deletions

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Substitutions

• A base-pair substitution is the replacement of
  one nucleotide and its partner with another pair
  of nucleotides
• Silent - changes a codon but codes for the same
  amino acid
• Missense - substitutions that change a codon for
  one amino acid into a codon for a different amino
  acid
• Nonsense -substitutions that change a codon for
  one amino acid into a stop codon

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Insertions and Deletions

• Insertions and deletions
• Are additions or losses of nucleotide pairs in a
  gene
• May produce frameshift mutations that will change
  the reading frame of the gene, and alter all
  codons downstream from the mutation.

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Overview

• Protein Synthesis/Gene Expression
• Transcription
• Initiation
• Elongation
• Termination
• Translation
• Initiation
• Elongation
• Termination
• Mutations
• Causes and rate
• Mutagens
• Physical
• Chemical
• Viral
• Point mutation
• Insertions and deletions
• Substitution