Transcript Gene to Protein

Gene to Protein
I.
What do genes do?
A. Garrod’s work
B. Beadle and Tatum
A.
Garrod-Alkaptonuria-1902
1. inborn error of metabolism
2. “unit factor” makes functional ferment
(A)
3. recessive (a) makes nonfunctional
ferment
4. AA normal Aa makes sufficient
5. aa homozygote makes no ferment
6. His work proved the link between
genetics and the production of proteins
B. Beadle and Tatum
Beadle and Tatum protocol
II.
Protein synthesis-an overview
A. Information flow
• 1. DNA->RNA->protein
• 2. the process involves two distinct classes of
molecules
• 3. nucleic acids with the monomers of the four bases
A, G, T, C
• 4. the proteins with their monomers of amino acids
• 5. each possess polymers that differ from other
polymers by the sequence of the monomers
• 6. Think of it in a language sense
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DNA->RNA is called transcription
a.
a scribe was an ancient who copied original texts
b.
DNA serves as the template from which mRNA is
manufactured
c.
the language is the same-different dialect
d.
will see that the RNA has a different nucleotide base
e.
uracil replaces thymine in the structure of RNA
f.
thymine possesses a methyl group that uracil lacks
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RNA->Protein is called translation
a.
in this process you are changing languages
b.
different script as well as vocabularly
c.
the order of the nucleotide sequence is being
used to direct the line up of the amino acids
in the polypeptide chain that will grow
d.
the site of this translation are the ribosomes
of the cell
B. An analogy might help
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Valuable cookbook
Copy recipe
Carry recipe copy to kitchen
Produce pasta fagiole
• A polypeptide 140 amino acids long would be coded for
by a mRNA ________ nucleotides long.
III. The genetic code
A. How long is a genetic word?
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codon
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the code is redundant but not ambiguous
B. Code is cracked in 1961 by Marshall Nirenberg
C. Nature of the code
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the code redundant
the code is not ambiguous
one codon never represents more
than one unique amino acid
there are punctuation codons in
the list
the start codon is always AUG
there are also termination codons
D. How is the code read?
• 1. nonoverlapping or overlapping?
• 2. a reading frame is established by the
presence of the start codon
• 3. from there on out, the nucleotides are read
every three
• 4. Thecatanddogranoutthe
• 5. as long as this string is read in the correct
reading frame there is a message
• 6. get out of the frame and it is just gibberish
E.
Genetic code evolved very early in life
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the genetic code is shared by all living forms of life
Wobble theory
a.
notice that in the genetic dictionary-most of the
specificity seems to reside in the first two letters of the
codon
b.
the third nucleotide of the codon seems to be less
important in determining which amino acid is being
specified
c.
when there is redundancy it resides in the location of
the third base pair
d.
believed that ancestrally there were sixteen amino
acids
e.
if there are only sixteen amino acids-codon needs to
be only two nucleotides long 42 = 16 unique
combinations
f.
it is believed that the third position served as a spacer
g.
with evolution need to expand into the third spacer position
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IV. RNA
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Differences between
DNA and RNA
1. DNA is double helix
2. RNA is a single
stranded molecule with
sugar phosphate
3. uracil replaces
thymine
4. RNA is a shorter
molecule
5. RNA can travel from
the nucleus to the
cytoplasm
B. Types of RNA-basically three
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messengerRNA (mRNA) is a copy of
the gene
ribosomalRNA (rRNA) produces a
portion of the ribosome
transferRNA (tRNA)
C. RNA production-Transcription
http://www.youtube.com/watch?v=WsofH466lqk
http://www.youtube.com/watch?v=5MfSYnItYvg
1. Initiation-beginning
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RNA polymerase is the enzyme of
transcription
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it attaches to the promoter of the gene in
question
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almost universal in eukaryotic
promoters is the TATA box
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found upstream from the start point
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a transcription factor binds to the
TATA segment before RNA polymerase
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transcription factors are proteins that
regulate the transcription of the gene
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they can either turn on or turn off
transcription by regulating accessibility to
the promoter by the RNA polymerase
2. Elongation stages
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RNA polymerase unwinds the DNA double helix
exposes 20 DNA bases at a time
serves as a platform for RNA-DNA nucleotide base pairing
can also only attach in the 5’->3’ direction
produces the chain at the rate of 60 nucleotides/sec
the RNA detaches from the RNA polymerase while the DNA goes
back into helix
g. multiple RNA polymerases can ride along the DNA transcribing
multiple copies of the gene in question
3. Chain termination
a. when the RNA polymerase
reaches the termination
sequence it releases both
the mRNA and the DNA
template
b. the release point on
eukaryotic mRNA is
downstream from the
actual termination segment
c. the cleavage point is also
the site for the attachment
of the polyAAA tail
D. Production of the eukaryotic mRNA produces a primary transcript
1. this primary transcript must be
modified before it leaves the
nucleus
2. RNA splicing
a. an average protein is
about 400 amino acids
b. therefore theoretically
about 1200 nucleotides are
needed for coding
c. eukaryotic genes are
usually much longer than
this
d. exons
e. introns
Some interesting facts about introns
• a. almost nonexistent in the prokaryotic world
• b. their frequency seems to increase in the more complicated
organisms
• c. sponges have fewer introns than do flatworms
• d. flatworms have fewer introns than do round worms
• e. as you climb the evolutionary tree-introns become more
ubiquitous
• f.
in eukaryotic genes, there can be tens to hundreds of introns
which are hundreds of nucleotides long
• g. is it fair to call introns junk DNA based on the above?
Some possible functions of introns
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safe zones for mutations
at a later time in the future the intron could become a functional exon
introns may regulate the transcription of the gene
splicing of different exons increases protein product
introns may represent past viral infections that are in a dormant state
3. Modification of ends
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5’ end of the molecule
a. first segment produced
b. receives a cap of GTP
c. seems to serve several functions
d. protects the end first produced from degradation
e. serves as a signal for the ribosome that this is where you
attach
f.
might serve as an energy source for the attachment of the
ribosome
the 3’ end of the mRNA receives a polyA tail
a. several functions
b. again protection from degradation
c. somehow signals for the export of the mRNA to the cytoplasm
d. might be used as a counter serving to control the life span of
the mRNA
Completed mRNA transcript ready for translation
V. The synthesis of proteins-translation
A. Overview
https://www.youtube.com/watch?v=5bLEDd-PSTQ
V.
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The synthesis of proteins-translation
A.
Overview
1. all of the amino acids needed
to produce proteins are stockpiled in
the cytoplasm
2. these amino acids are either
synthesized by the body or the amino
acids are absorbed from the diet
3. a recipe arrives in the
cytoplasm containing the order that the
amino acids should be assembled in
4. the order is present in the
sequence of codons possessed on the
mRNA
6. a ribosome attaches to the
mRNA, runs down the mRNA, links
amino acids together according to the
instructions
• 7. key to the process is that there
are specific tRNA’s that are unique for
each amino acid
• 8. the tRNA’s loaded with a specific
passenger are floating around the
cytoplasm
• 9. by random molecular collisions
exposed codons on the surface of the
ribosome attach to corresponding
anticodons on the surface of the tRNA
• 10. peptide bonds can be formed
between the two adjacent amino acids
• 11. the ribosome can progress along
the mRNA to the next codon
B. tRNA’s
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produced in the nucleus like all RNA
only about 80 nucleotides long
Internal complementary base pairing
it folds into a structure that looks a bit like a cloverleaf
two very important regions of the tRNA
the anticodon sticks out of one side and the 3’ end has a specific site for the
attachment of an amino acid
C.
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Aminoacyl-tRNA synthetases
this process occurs in the
cytoplasm of the cell
specificity between each amino
acid and the tRNA that delivers
it to the site of protein
synthesis
this depends upon the
appearance of specific
enzymes that do a job
aminoacyl-tRNA synthetase
there are twenty different
varieties of this enzyme-each
specific in its shape
the synthetase attaches the
amino acid to the tRNA that is
then free to wander around
until it joins a ribosome
who makes the aminoacyltRNA synthetases?
D. Ribosomes
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1. composed of two subunits2. the ribosomal subunits only
are found together while they
are assembled on a mRNA
3. the total weight of a
ribosome is about 60% RNA
and 40% protein
4. the rRNA is transcribed in
the nuclear region known as
the nucleolous
5. literally thousands of
ribosomes in a cell, rRNA is
by far the most common RNA
possessed by a cell
6. the ribosomes are
assembled in the nucleolus
from rRNA and proteins
imported from the cytoplasm
7. the completed subunits are
then exported through the
nuclear pores and distributed
throughout the cytoplasm by
the ER
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Notice the three sites on the ribosome:
a. P
b. A
c. E
The ribosomes of prokaryotes and
eukaryotes are a bit different
a. function in much of the same way
b. structures are a little different as are their
chemistries
c. antibiotics make use of this subtle
difference in the manner in which they work
d. some antibiotics can block the
translation of bacterial genes while not
affecting the eukaryotic cell
the two parts of the ribosome
a. the smaller subunit keeps the mRNA in a
linear sequence allowing docking of the
tRNA’s
b. it also moves along the mRNA like a
train moving along tracks
c. the larger subunit appears to be the part
of the molecule which helps the peptide
bond to form
polyribosome
E. Building a polypeptide-three simple steps
• 1. chain initiation 2. elongation 3. termination
1. Chain initiation
• a. small subunit of ribosome
attaches upstream to the start
codon
• b. initiator tRNA with methionine
recognizes the start codon AUG
• c. through the use of GTP, the
large subunit then attaches to
finish the formation of the
translation initiation complex
• d. notice that the A site on the
ribosome is open allowing the next
step of translation to begin
2. Chain elongation-grows by 10
amino acids per second
3. Chain termination
4. Polyribosome
F. Signal polypeptides-proteins for export
VI. Mutations-the ultimate source of
variation-will only talk about point mutations
A. Substitution-one nucleotide is substituted for a
second
1. silent mutation due to
redundancy of the code
2. missense mutation
a. One amino acid is exchanged
for a second
b. Sickle cell anemia is an
example
Change in primary structure
Heterozygote advantage
3. Nonsense mutation-premature
termination of translation
B. Insertion-deletion mutations cause a frame shift
C. Which is the worse?