Transcription and Translation

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Transcription and Translation

• Tuesday, August 5

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From Genes to Proteins

• The genetic material, DNA, contains discrete
  units, genes, that specify certain traits
• How do genes confer specific phenotypes?
• The function of a gene is to dictate the
  production of a specific protein or enzyme
• The enzyme catalyzes a certain chemical reaction
  in the cell
• Differences or mutations in these genes cause
  different or defective proteins leading to a
  given phenotype

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One gene one enzyme hypothesis (Beadle and
Tatum)

• Studying the mold Neurospora
• Wild type mold can make its own amino acids
• Treating mold with X-rays damages DNA, causing
  mutations in specific genes
• Mutants can grow on complete media
• Determined mutant gene by growing mutant species
  on minimal media plus one of the 20 amino acids
• Found 3 classes of mutants that had mutations in
  synthesizing arginine

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Figure 17.1 Beadle and Tatums evidence for the
one gene-one enzyme hypothesis
Each mutant was defective in a single gene and
lack a specific enzyme
One Gene One Protein
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nucleic acids
amino acids
DNA
Protein
RNA
Transcription
Translation
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Figure 17.2 Overview transcription and
translation
mRNA
DNA provides the template for RNA synthesis. A
copy of a gene is produced called a messenger RNA
(mRNA) which carries the instructions from DNA to
the protein synthesizing machinery.
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Figure 17.2 Overview transcription and
translation
mRNA provides the template for protein synthesis.
Nucleic acids are translated into amino acids by
ribosomes.
In a prokaryotic cell, the two steps occur
simultaneously since there is no nucleus to
separate the processes.
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Figure 17.2 Overview transcription and
translation
In a eukaryotic cell, the nucleus separates the
DNA from the ribosomes in the cytoplasm. mRNA
synthesized in the nucleus is called the primary
transcript.
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Figure 17.2 Overview transcription and
translation
The primary transcript undergoes RNA processing
steps, which modifies it in certain ways. The
mRNA is then translocated from the nucleus to the
cytoplasm.
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Figure 17.2 Overview transcription and
translation
Once in the cytoplasm, ribosomes bind to the mRNA
and synthesize the specified protein.
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The Genetic Code

• How does the mRNA nucleotide sequence dictate the
  amino acid sequence of proteins during
  translation?
• 4 nucleotides specify 20 amino acids
• Triplets of nucleotides could code for single
  amino acids
• 43 64 possible code words (amino acids)
• Triplet codon genetic instructions for a
  polypeptide chain are written in the DNA as a
  series of three-nucleotide words
• example AGT Serine
• DNA is double stranded which strand is
  transcribed and translated?
• Template or Coding strand

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Figure 17.3 The triplet code
The transcript is complementary to the coding
sequence. RNA uses uracil (U) in place of
thymine (T) Codons are read in the 5 ? 3
direction ACC ? UGG ? Trp 300nt gene 100 aa
protein
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Figure 17.4 The dictionary of the genetic code
The genetic code was solved when a synthetic
oligonucleotide of uracil produced a chain of
phenylalanine amino acids. The genetic code is
redundant tends to occur in the third aa.
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Features of the Genetic Code

• DNA encodes a sequence of nonoverlapping base
  triplets codons
• AUGACGAAGAGGGGAUAA
• Read AUG ACG AAG AGG GGA UAA
• Not read AUGACGAAG AUG UGA GAC ACG
• Reading Frame codons must be read in the correct
  groupings
• AUGACGAAGAGGGGAUAA
• Read AUG ACG AAG AGG GGA UAA
• Not read UGA CGA AGA GGG GAU

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Genetic Code is Universal

• This genetic code is shared by organisms from the
  simplest bacteria to the most complex animals and
  plants
• This allows the transplanting of DNA from one
  species to another
• Human genes can be inserted into bacteria to
  produce proteins for medical uses
• The universality suggests that the genetic code
  must have evolved early in the history of life

Figure 17.5 A tobacco plant expressing a firefly
gene. The plant is programmed to produce a
firefly protein by transplanting the DNA
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Figure 17.6 The stages of transcription
initiation, elongation, and termination
Transcription is INITIATED at special sequences
called PROMOTERS where an enzyme, RNA polymerase
binds. Prokaryotic cells have one RNA
polymerase. Eukaryotic cells have 3 (I, II, and
III) RNA pol II - synthesizes mRNA
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Figure 17.6 The stages of transcription
initiation, elongation, and termination
The two strands of DNA are unwound and separated.
RNA polymerase initiates synthesis at the start
codon on the template (coding) strand.
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Figure 17.6 The stages of transcription
initiation, elongation, and termination
upstream
downstream
DNA reforms a double helix in the wake of
transcription
The transcript is ELONGATED as the RNA pol moves
downstream, unwinding the DNA and adding RNA
molecules complementary to the template.
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Figure 17.6 The stages of transcription
initiation, elongation, and termination
Transcription is TERMINATED when the RNA pol
translates a terminator sequence. The RNA
transcript is released and the polymerase
detaches from the DNA
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Transcription Initiation
Figure 17.7

• Promoters
• Transcriptional start site (AUG)
• Binding sequence for RNA pol (TATA box)
• Located about 25 nucleotides from the start site
• Designates the template strand
• Initiation complex
• Eukaryotic transcription factors mediate the
  binding of RNA pol to the promoter
• These factors unwind and separate the DNA and RNA
  synthesis begins

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Transcription Elongation

• Elongation occurs in the 5 ? 3 direction
• As RNA pol moves downstream, DNA unwinds about
  10-20 bases at a time
• Transcription rate 60 nucleotides/sec
• One mRNA can be transcribed simultaneously be
  several RNA pols, increasing the amount of mRNA
  produced

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Transcription Termination

• The transcribed terminator sequence (RNA) is the
  stop signal
• Prokaryotes terminate immediately following this
  signal
• Eukaryotes proceeds hundreds of nucleotides past
  this signal (AAUAAA)
• About 10-35 nts past the signal, the mRNA is
  cleaved

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RNA processing addition of the 5? cap and
poly(A) tail

• 5 cap modified guanine (G)
• Protects mRNA from degradation
• Binding site for ribosomes in the cytoplasm
• 3 poly A tail 50-250 adenines (A)
• Protects mRNA from degradation
• Binding site for ribosomes
• Aids in transport of mRNA to cytoplasm

Fig 17.8
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RNA Splicing

• Splicing
• RNA transcripts have long stretches of noncoding
  regions (introns) interspersed between coding
  regions (exons)
• Introns are cut out and exons are joined together
• Spliceosome
• Composed of snRNPs and other proteins
• Small nuclear ribonucleoproteins (snRNPs) bind to
  splice sites (short sequences at the end of
  introns)

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Figure 17.10 The roles of snRNPs and
spliceosomes in mRNA splicing
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Functionality of Introns

• Alternate splicing
• Different splicing in different cells or
  organisms can include or exclude certain exons
• Genes can give rise to two or more different
  polypeptides
• Functional domains
• Proteins often have discrete structural and
  functional domains
• Introns provide a mechanism for recombination
  between exons of different genes
• Exon shuffling could lead to new proteins with
  novel combinations of functions

Fig. 17.10
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nucleic acids
amino acids
DNA
Protein
RNA
Transcription
Translation
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Translation Overview

• Interpreting a genetic message series of codons
  along an mRNA
• Interpreter transfer RNA (tRNA)
• Transfers amino acids from cytoplasm to ribosome
• Machinery ribosome
• Adds aa from tRNA to the growing polypeptide
  chain
• As mRNA moves through a ribosome, codons are
  translated into amino acids

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Figure 17.12 Translation the basic concept

• Each type of tRNA linnks a particular mRNA codon
  with a particular amino acid
• One end binds a specific amino acid
• Other end is the anticodon, nucleotide triplet
  that is complementary and binds to the codon on
  the mRNA transcript
• AAG binds UUC in the mRNA and adds Phe

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Figure 17.13a The structure of transfer RNA
(tRNA)

• The secondary structure of tRNA looks like a
  cloverleaf
• There are three stem-loop structures
• The anticodon is specific for each tRNA
• AAG binds UUC in the mRNA and adds Phe

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Figure 17.13b The structure of transfer RNA
(tRNA)
The tRNA folds into an L shape 3D
structure Anticodons are written 3 ? 5 to
align with the mRNA codon.
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Wobble Theory

• If one tRNA existed for each codon that specifies
  an amino acid, there would be 61 tRNAs there
  are 45.
• The base pairing rules are relaxed for the 3rd
  amino acid in a codon
• U can bind with either A or G in the mRNA
• The redundancy of the genetic code permits this
  wobble
• UUA and UUG both code for leucine, so a U in the
  anticodon will be able to bind either codon and
  translate it to leucine

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Figure 17.14 An aminoacyl-tRNA synthetase joins
a specific amino acid to a tRNA
There are 20 different enzymes that catalyze the
addition of the 20 different amino acids to its
specific tRNA. These enzymes are termed
aminoacyl-tRNA synthetases. The addition of the
amino acid requires energy (ATP) and this process
activates the tRNA for delivery of the amino acid
to the ribosome for attachment to the growing
peptide chain.
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Figure 17.0 Ribosome
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Figure 17.15 The anatomy of a functioning
ribosome

• Ribosomes complex of proteins and ribosomal RNA
  (rRNA)
• Large subunit
• Binding sites for tRNAs
• E (exit site)
• P (peptidyl-tRNA site)
• A (aminoacyl-tRNA site)
• Small subunit
• Binding site for mRNA
• Ribosome holds the mRNA and the tRNA in close
  proximity
• Addition of the new aa to the carboxyl end of the
  chain
• Catalyze formation of peptide bond

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Figure 17.17 The initiation of translation

• Small subunit binds to the mRNA through a special
  binding site that recognizes a specific sequence
• An initiator tRNA binds which carries Met
• The large subunit binds, placing the tRNA in the
  P site
• Energy (GTP) and initiation factors are required
  for this assembly

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Figure 17.18 The elongation cycle of translation
Incoming tRNA binds the codon in the A site
The ribosome catalyzes the formation of a peptide
bond
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Figure 17.18 The elongation cycle of translation
Incoming tRNA binds the codon in the A site
The tRNA in the P site is translocated to the E
site where it is released
The tRNA in the A site is translocated to the P
site, taking the mRNA along
The ribosome catalyzes the formation of a peptide
bond
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Figure 17.19 The termination of translation

• Elongation continues until a stop codon in the
  mRNA reaches the A site of the ribosome
• UAA, UAG, UGA
• A release factor protein binds directly to the
  stop codon in the A site and causes the addition
  of water, which hydrolyzes and releases the
  polypeptide chain
• The assembly dissociates

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Figure 17.20 Polyribosomes

• An mRNA molecule is translated by several
  ribosomes simultaneously
• The strings of ribosomes are called polyribosomes
• Many copies of a protein can be synthesized
  quickly
• A single ribosome can make a protein in less than
  a minute

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Targeting Proteins

• Once proteins are formed, they must reach their
  ultimate destination
• Cytosolic proteins
• synthesized on FREE ribosomes in the cytoplasm
• ER, Golgi, lysosomal, plasma membrane and
  secreted proteins
• synthesized on ribosomes BOUND to the cytosolic
  side of the ER
• Protein synthesis starts in the cytosol and
  continues there unless the protein itself directs
  the ribosome to the ER
• Proteins contain a signal peptide
• 20 aa near the leading end of the polypeptide
• Recognized by a signal-recognition particle (SRP)
• Brings the ribosome to a receptor in the ER
  membrane

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Figure 17.21 The signal mechanism for targeting
proteins to the ER

• SRP binding causes synthesis to stop
• Once the ribosome is set up at the ER membrane,
  the SRP leaves and synthesis continues
• The growing chain is translocated through a pore
  in the ER membrane
• An enzyme cleaves off the signal sequence

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Table 17.1 Types of RNA in a Eukaryotic Cell

• RNA has many important roles
• Structural
• Informational
• Catalytic
• RNA can hydrogen bond to
• other nucleic acids (RNA/DNA)
• itself, forming specific 3D shapes

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Figure 17.22 Coupled transcription and
translation in bacteria
Prokaryotes vs. Eukaryotes

• Transcription
• Different RNA polymerase
• Eukaryotes depend on transcriptions factors
• Termination different
• Eukaryotes have RNA processing steps
• Translation
• Different ribosomes
• Eukaryotes target proteins to appropriate
  compartments
• Compartmentalization

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Mutations in the Genetic Code

• Point mutations
• Base pair substitution
• The replacement of a nucleotide (wildtype) and
  its partner with another nucleotide (mutant)
• Silent mutation does not affect aa due to
  redundancy
• CCG mutated to CCA ? both still code for glycine
• Missense mutation codes for a different aa
• Nonsense mutation codes for a stop codon
• Base pair insertions or deletions
• Additions or loses of nucleotides
• Frameshift mutation
• Alter the reading frame of a genetic message

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Figure 17.24 Categories and consequences of
point mutations Base-pair substitution
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Figure 17.24 Categories and consequences of
point mutations Base-pair insertion or deletion
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Figure 17.23 The molecular basis of sickle-cell
disease a point mutation
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Causes of Mutations

• Spontaneous mutations
• Errors during DNA replication, repair, or
  recombination
• Mutagens
• High-energy radiation
• UV rays
• X-rays
• Base analogues
• Chemical mutagens
• carcinogens

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Figure 17.25 A summary of transcription and
translation in a eukaryotic cell