Nerve activates contraction

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CHAPTER 11 How genes are expressed
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What is RNA?

• Single stranded nucleic acid
• Base pairs A-U, C-G
• Contains ribose sugar
• Much shorter strand than DNA

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Gene product is a polypeptide
PROTEIN
The information from DNA is copied into an
intermediate molecule known as
RNA Transcription
The information in RNA is ultimately
transferred into a protein by a process
called Translation
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What exactly is a gene?

• Specific sequence of nucleotides on a chromosome
  that has defined start/stop regulatory regions.

A gene will have precise locations with specific
roles in expression. Expression of our genes are
highly regulated!
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Transcription

• DNA provides a template for the synthesis of a
  complementary RNA strand.
• DNA has sense and anti-sense strand.
• RNA polymerase reads the anti-sense strand. So
  mRNA basically is the sense strand, with U
  instead of T.

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• Promoter region 1) site on gene where
  transcription factors bind.
• 2) RNA polymerase begins transcription

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What is a transcription factor?

• Proteins that regulate the expression of a gene.
• Activate or inhibit gene expression
• RNA pol. recognizes T.Fs binds when the
  proteins are there.

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Gene Regulation
TFs can have 1)DNA binding domain, 2)domain to
bind to other TFs ( some Only work in pairs)
3) domain to bind w/ RNA pol.
Mutations in JUN/FOS have been shown in cancer
cells.
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• RNA polymerase
• 1) Separates the DNA strands at the appropriate
  point
• 2) Binds the RNA nucleotides as they base-pair
  along the DNA template.

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polymerase

• RNA pol. unwinds 10 bases at a time.
• New RNA molecule peels away.
• DNA re-anneals

mRNA
DNA
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• Terminator sequence present at the end of the
  transcription unit.
• The terminator signals the end of transcription.
• Transcription proceeds until RNA polymerase
  transcribes a terminator sequence in the DNA.

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• Termination
• RNA polymerase continues adding nucleotides
  until it identifies terminator sequence as
  AAUAAA.
• It transcribes the termination sequence and goes
  PAST the termination.
• Then, pre-mRNA is cut from the enzyme.

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Animation transcription

• http//images.google.com/imgres?imgurlhttp//vcel
  l.ndsu.nodak.edu/christjo/vcell/animationSite/tra
  nscription/Stills/1065.jpgimgrefurlhttp//vcell.
  ndsu.nodak.edu/christjo/vcell/animationSite/trans
  cription/poll2.htmh480w640sz45tbnidu14AXK7
  UcnwJtbnh101tbnw135hlenstart4prev/image
  s3Fq3Dtata2Bbox26hl3Den26lr3D
• Animation Translation
• http//images.google.com/imgres?imgurlhttp//vcel
  l.ndsu.nodak.edu/christjo/vcell/animationSite/tra
  nscription/Stills/1065.jpgimgrefurlhttp//vcell.
  ndsu.nodak.edu/christjo/vcell/animationSite/trans
  cription/poll2.htmh480w640sz45tbnidu14AXK7
  UcnwJtbnh101tbnw135hlenstart4prev/image
  s3Fq3Dtata2Bbox26hl3Den26lr3D
• Animation Lac Operon (inducible system)
• http//images.google.com/imgres?imgurlhttp//vcel
  l.ndsu.nodak.edu/christjo/vcell/animationSite/tra
  nscription/Stills/1065.jpgimgrefurlhttp//vcell.
  ndsu.nodak.edu/christjo/vcell/animationSite/trans
  cription/poll2.htmh480w640sz45tbnidu14AXK7
  UcnwJtbnh101tbnw135hlenstart4prev/image
  s3Fq3Dtata2Bbox26hl3Den26lr3D

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pre-mRNA undergoes 3 modifications

• 5 cap
• at the 5 end, a modified form of guanine is
  added.
• Functions
• This helps protect mRNA from hydrolytic enzymes.
• It also functions as an attach here signal for
  ribosomes.

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• 2) poly(A) tail
• At the 3 end, an enzyme adds 50 to 250 adenine
  nucleotides
• Functions
• Poly A tail inhibits breakdown of mRNA
• facilitating ribosome attachment
• seems to facilitate the export of mRNA from the
  nucleus.

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3) Introns spiced out

• Eukaryotic pre-mRNA undergoes splicing.
• Noncoding segments, introns, that are present
  between coding regions are removed or spliced.
• Coding sequences or exons are joined to form mRNA

EXON
INTRON
EXON
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• Genes are made up of coding and non-coding
  regions called exons and introns.

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• RNA splicing thus creates a mRNA molecule with a
  continuous coding sequence.
• It retains Cap and Poly A tail
• The process of splicing is facilitated by a
  ribonucleoprotein called a spliceosome

mRNA with just exons
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Recap Transcription

• Begin at promoter region (TATA box) where
  transcription factors RNA pol. bind
• RNA pol. unwinds and adds nucleotides to form a
  strand of RNA
• This strand of RNA undergoes 3 modifications 1)
  5 G-cap 2) 3 poly A tail 3) introns spliced
  out.
• Now we have a mature mRNA, ready to leave nucleus
  to cytoplasm.

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Translation RNA to Protein

• the information contained in the order of
  nucleotides in mRNA
• is used to determine the amino acid sequence of a
  polypeptide.

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Central dogma of life

• Remember! DNA ? RNA ? protein.
• How is the information from an mRNA molecule
  translated into a protein molecule?
• With the help of genetic code!

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• Triplets of nucleotide bases code for a specific
  amino acid.
• These triplets are called codons
• Your genes are a series of codons linked together
  that code for a series of amino acids that will
  be linked together!

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• During translation, blocks of three nucleotides
  present in mRNA, codons,
• are decoded into a sequence of amino acids.
• Leading to the formation of protein

Fig. 17.3
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• Only 20 amino acids, but there are 64 possible
  combinations for codons.
• 61 out of the 64 code for proteins
• The other three
• Stop codons (3) do not code for amino acids,
  signal the termination of translation.
• Start codon (1)AUG (also codes for methionine)
• also indicates the start of translation.

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• The genetic code is redundant.
• I.e. several codons for an amino acid.
• Any given codon represents only one amino acid.
• Codons synonymous for the same amino acid often
  differ only in the third codon position.

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Universal applicability of genetic code

• The genetic code is nearly universal!
• Shared code by organisms from bacteria to
  mammals.
• Therefore, you can synthesize certain human
  proteins in bacteria
• Such as insertion of insulin gene.

This tobacco plant is expressing a transpired
firefly gene
Fig. 17.5
break
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Overall view of Translation
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Transfer RNA (tRNA)

• transfers amino acids from the cytoplasms pool
  to a ribosome.
• A tRNA molecule consists of a strand of 80
  nucleotides that folds back on itself
• to form a three-dimensional structure
• resembling clover leaf

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• It includes a loop containing the anticodon and
• an attachment site at the 3 end for an amino
  acid.

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• Each tRNA triplet, an anticodon, is specific for
  an amino acid.
• The anticodon base-pairs with a complementary
  codon on mRNA
• Eg) phenyalanine
• mRNA codon UUC
• tRNA anti-codon AAG

PHE
AAG
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• Ribosomes
• Each ribosome has a large and a small subunit.
• These are composed of
• proteins and
• ribosomal RNA (rRNA), the most abundant RNA in
  the cell.

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• 1 binding site for mRNA
• Three binding sites for tRNA molecules.
• The P site holds the tRNA carrying the growing
  polypeptide chain.
• The A site carries the tRNA with the next amino
  acid.
• E site is the site where discharged tRNAs leave
  the ribosome.

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Translation can be divided into three stages

• Initiation
• Elongation
• termination
• All three phase require protein factors that
  help in the translation process.
• require energy in the form of GTP.

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• Initiation brings together
• mRNA, a tRNA with the first amino acid, and the
  two ribosomal subunits.
• Initiation factors bring in the large subunit
  such that the initiator tRNA occupies the P site.

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• Elongation
• What needs to happen here
• 1) recognize the codon
• 2) form a peptide bond
• 3) move the newly attached amino acid over, so we
  can add a new one (translocation).
• REPEAT

Next tRNA Goes here
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• Codon Recognition
• 1) mRNA codon under the A site of ribosome
• 2) corresponding anticodon of tRNA carrying
  appropriate amino acid.

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New peptide bond

• peptide bond formation
• an rRNA molecule of ribosome catalyzes the
  peptide bond.
• The new amino acid comes in at the A site,
  elongation at the P site.
• Empty tRNA leaves at E site.

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• The empty tRNA that had been in the P site is
  moved to the E site.
• This is called translocation
• The tRNA is recycled.
• Amino acids are added on this way, one by one
  until it hits the stop codon.

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• Termination occurs when one of the three stop
  codons reaches the A site.
• A release factor binds to the stop codon
• Releases polypeptide from tRNA at P site.
• the translation complex disassembles.

Fig. 17.19
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• The new amino acid chain then folds into its
  functional 3D shape.

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There are two types of ribosomes which
participate in protein synthesis

• Bound ribosomes
• Bound ribosomes are attached to the cytosolic
  side of the endoplasmic reticulum
• They synthesize secretory proteins to be secreted
  out or to be inserted into endomembranous system

• free and
• Free ribosomes are suspended in the cytosol and
• synthesize proteins that reside in the cytosol

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• translation animation
• ??
• Transcription
• http//www.stolaf.edu/people/giannini/flashanimat/
  molgenetics/transcription.swf
• Translation
• http//www.stolaf.edu/people/giannini/flashanimat/
  molgenetics/translation.swf

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So if all goes well..
DNA is ok. RNA trancription went well.
tRNA reading goes well/ Amino acid Bonds
correct. Protein folds Into correct
conformation,
You have A functional New protein!
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Point mutations can affect protein structure and
function

• Mutations are changes in the genetic material of
  a cell
• A chemical change in just one base pair of a gene
  causes a point mutation.
• E.g.Sickle cell anemia
• If these occur in gametes or cells producing
  gametes,
• they may be transmitted to future generations.

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• Sickle-cell disease is caused by a mutation of a
  single base pair in the gene that codes for one
  of the polypeptides of hemoglobin.
• A change in a single nucleotide from T to A in
  the DNA template leads to an abnormal protein.

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• Mutations can occur spontaneously!
• mutations can occur during DNA replication DNA
  repair, and not be fixed.
• These are called spontaneous mutations.

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• Mutations can be caused by 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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Free radicals

• These are formed in our bodies during cellular
  respiration and by our immune system.
• Atom or molecule with unpaired electronhighly
  unstable.
• It will bind with any molecule to become stable.
  This haphazard binding can destroy biological
  molecules eg) phospholipids in our cell
  membranes.
• Antioxidants Vitamin E, Vitamin C soak up
  these free radicals.

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Base Pair Substitutions
1) silent mutations The codon still codes for the
same a.a. 2) neutral mutations Change in codon
does change a.a. ,but does not affect protein
funtion. 3) Missense mutation Change in codon
changes aa which has an harmful effect on protein
function
4) Nonsense mutation Mutation leads to a stop
codon. Translation stops prematurely, Get
non-functional protein.
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Frameshift mutationsInsertions and deletions

• If you add an extra nucleotide to the sequence of
  DNA, you get an insertion error.
• If you delete a nucleotide, you get a deletion
  error.
• These mutations are much worse than
  substitutions. Why is this?

Example of deletion
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Because you change the reading frame

• Your gene is a series of codons that are read in
  one direction.
• If you add or delete one nucleotide, you shift
  the entire reading frame over, so any protein
  coded for after the mutation will be wrong.
• Called a FRAMESHIFT mutation.

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DNA repair