Storage and use of genetic information

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Storage and use of genetic information

• The genetic code
• Three bases (in a row) specify an amino acid
• Transcription
• The synthesis of a mRNA, complementary to one of
  the DNA strands, containing the genetic code
• Translation
• Proteins and rRNAs in the ribosome along with
  tRNAs translate the genetic code into proteins.
• Post-translational modification
• Proteins are altered after synthesis

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The Genetic Code

• Four bases taken how many at a time? Need to code
  for 20 different amino acids.
• Each base 1 amino acid only 4
• Every 2 bases 1 a.a. 16 combinations, 4
  short.
• Every 3 bases 64 combinations, enough.
• Every 3 bases of RNA nucleotides codon
• Each codon is complementary to 3 bases in one
  strand of DNA
• Each codon (except for T ?U switch) is the same
  as 3 bases in the other DNA strand.

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More about the Genetic Code

• The code is
• Unambiguous each codon specifies 1 amino acid
• Degenerate a particular amino acid can be coded
  for by several different codons.
• Ordered similar codons specify the same amino
  acid.
• Commaless, spaceless, and non-overlapping each
  3 bases is read one after the other.
• Punctuated certain codons specify start and
  stop.
• Universal by viruses, both prokaryotic domains,
  and eukaryotes (except for some protozoa,
  mitochondria).

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The Genetic Code-2
http//www.biology.arizona.edu/molecular_bio/probl
em_sets/nucleic_acids/graphics/gencode.gif
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Wobble

• Cricks Wobble Hypothesis
• The code is ordered
• The first 2 positions are more important
• When lining up with the anticodon of the tRNA,
  the third position doesnt bind as tightly, thus
  a looser match is possible.
• Because of this flexibility, a cell doesnt need
  61 different tRNAs (one for each codon).
• Bacteria have 30-40 different tRNAs
• Plants, animals have up to 50.

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Colinearity

• Archibald Garrod (1908) determined that genes
  must code for enzymes by studying metabolic
  diseases (black urine). But what relationship?
• Beadle and Tatum (1941) one gene one enzyme,
  there is a one to one correspondence between DNA
  and protein
• Mutation in DNA changes nucleotide sequence,
  changes amino acid sequence in protein.
• Not completely true in eukaryotes because of
  introns

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The code is colinear with protein sequence

• Bacteriophage MS2
• An RNA virus with 3 genes, including a viral coat
  protein.
• Amino acid sequence of coat protein determined,
  1970
• Sequence of coat protein gene determined, 1972
• Correspondence between codons and amino acids
  exactly as predicted.

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Ribosome structure
Large and small subunits. Eukaryotic 60S 40S
80S Prokaryotic 50S 30S 70 S
Large subunit 2 -3 rRNAs and many proteins Small
subunit 1 rRNA and many proteins
http//www.emc.maricopa.edu/faculty/farabee/BIOBK/
ribosome.gif
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Size in Svedbergs

• S is a unit based on ultracentrifugation
• What affects how fast particles move toward the
  bottom of the tube?
• Mass, density, and shape (which affects friction)
• Example supercoiled DNA moves faster than
  relaxed DNA.
• Because of these factors, S units are not
  additive
• 50S 30S prokaryotic subunits make a 70S
  ribosome.

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Components of ribosomes

• Multiple copies exist of ribosomal genes
• Def. of gene extended to DNA that codes for
  rRNA
• Multiple copies means moderately repetitive DNA
• Cells can crank out lots or rRNA (and r-proteins)
• Transcription results in RNA requiring processing
• Pre-RNA cut into rRNAs (and tRNAs)
• rRNA NOT just structural are ribozymes, carry
  out the actual protein synthesis.

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About tRNAs-1

• Coded for by tRNA genes
• Post-transcriptional modification
• tRNAs have some bases changed
• tRNAs interact with rRNA during protein synthesis
• tRNAs must have amino acid attached
• Enzymes aminoacyl tRNA synthetases
• 20 different enzymes, one for each aa.
• Enzymes recognize shape of tRNA

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About tRNAs-2

• tRNA structure must be highly conserved
• Must be folded so that both the synthetases
  recognize it, and it fits properly on the
  ribosome.
• Errors in translation result in bad proteins
  mutations in tRNA genes are selected against.

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tRNA
3D structure The familiar loops of the 2D
structure are labeled.
3 end Attaches to amino acid.
Decoder end Complementary to codon.
hto-b.usc.edu/cbmp/2001/ tRNA/trna20s1.jpg
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Translation

• mRNA provides message to be translated.
• Ribosomes functional workbench for synthesis.
• tRNA bring aa to ribosome, decode mRNA.
• Aminoacyl tRNA synthetases enzymes that attach
  amino acids to tRNAs.
• Protein factors help move process along
  initiation, elongation, and termination.
• Process is similar, but different between
  prokaryotes and eukaryotes.

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Initiation and Termination of protein synthesis

• AUG is always the first codon (initiator codon)
• Establishes an open reading frame (ORF)
• Ribosome begins synthesis with a methionine
• In bacteria, it is N-formylmethionine (fMet)
• After synthesis , either formyl group is removed
  or entire fMet is removed (Met in eukaryotes)
• Three codons serve as termination codons
• UGA, UAG, UAA any one can be a stop signal
• Do NOT code for an amino acid
• Cause translation to end protein is completed

formyl
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Translation-1

• Initiation
• Small subunit, mRNA, met-tRNA, IFs, GTP
• mRNA sequence for binding to ribosome needed
• prokaryotes Shine-Delgarno
• Eukaryotes Cap and Kozak sequence
• (GCC)RCCATGG where R is a purine
• First tRNA is fMet-tRNA in prokaryotes
• IFs are protein Initiation Factors
• GTP needed for energy
• When all have come together, Large subunit added

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Translation-2

• Ribosome has 3 sites
• AA site where tRNA-aa first sits in
• P site where tRNA with growing peptide sits
• E or Exit, site transiently occupied by used tRNA
• Elongation, with help of EFs and GTP
• tRNA with new aa sits in A site
• Stays in A site if anticodon on tRNA is
  complementary to codon on mRNA.
• tRNA in P site transfers growing chain to new aa
• Catalyzed by rRNA
• Ribosome moves relative to mRNA and tRNAs
• tRNAs now in new sites, new codon lined up

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Ribosome schematic
http//staff.jccc.net/pdecell/proteinsynthesis/tra
nslation/elongation12.gif
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Translation-3

• Termination
• When stop codon is in A site, no tRNA binds
• GTP-dependent release factor (protein) removes
  polypeptide from tRNA in P site. All done.
• Ribosomal subunits typically dissociate.
• Do a Google Search for translation animation
• Many hits. Note presence, absence of E site
• Note shape of ribosomes
• Note whether role of rRNA in catalysis is shown

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Nonsense mutations and suppressors

• A mutation may change a normal codon to a stop
  codon protein synthesis ends prematurely.
  (nonsense mutation)

A second mutation can cure the original a
suppressor. If the gene for a tRNA is mutated
in the anticodon so that the stop codon is now
read by the tRNA.
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Polysomes and Polycistronic mRNA

• In eukaryotes, when mRNA enters the cytoplasm,
  many ribosomes attach to begin translation. A
  mRNA w/ many ribosomes attached polysome.
• In eukaryotes, the mRNA for a single gene is
  processed and translated in prokaryotes, mRNA
  can be polycistronic, meaning several genes are
  on the same mRNA and are translated together
• With no nucleus, translation can begin in
  prokaryotes before transcription is over.

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Polysomes
Multiple ribosomes attach to the mRNA and begin
translating. Strings of ribosomes can be seen
attached to the mRNA.
http//opbs.okstate.edu/petracek/Chapter202720F
igures/Fig2027-29b-bottom.GIF www.cu.lu/labext/rc
ms/ cppe/traducti/tpoly.html
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Eukaryotic, prokaryotic differences

• mRNA lifetime is different
• Cap, tail, introns in eukaryotes
• Shine-Delgarno vs. Kozak sequence cap
• Size of ribosomes 70S vs. 80S
• fMet-tRNA vs. Met-tRNA
• Eukaryotic attachment of ribosomes to ER
• Polypeptides extruded through tunnel in large
  subunit, directly into ER

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Proteins vs. polypeptide

• Common usage
• Polypeptide is a string of amino acids
• Doesnt imply function
• Doesnt imply 3D shape
• Protein implies functionality and 3D shape
• Thus a protein can have a quaternary structure
  and be made of several different polypeptides.

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Review of protein structure
String of amino acids, covalently attached by
peptide bonds directional (N terminus, C
terminus).
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Primary structure
The particular amino acids and the order that
they are in.
20 different amino acids connected by peptide
bonds 100-1000 amino acids in a peptide chain.
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Secondary structure
Amino acid chain twists in space, held in place
by hydrogen bonds. Forms alpha helix or beta
pleated sheet.
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Tertiary structure
3-D folding of the protein chain in space the
shape is determined from the primary structure
and from the secondary structure (which itself
depends on the primary structure. The primary
structure depends on the info encoded in the DNA
Protein structure slides from
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Quaternary structure
Individual polypeptide chains aggregate to form a
single functional unit. Individual polypeptides
(protein subunits) may be switched out to give
the multipart protein a different function or
specificity.
Many proteins that act on DNA or RNA have a
quaternary structure.
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Domains
Different exons actually code for parts of
proteins that fold into discrete areas. May be
involved in evolution of proteins and their
functions.