Chapter 13: From gene to protein
Outline
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Metabolic defects provided evidence that genes specify
proteins
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Transcription and translation are the two main steps from
gene to protein
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The genetic code
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Translation is RNA-directed synthesis of a polypeptide
o Ribosomes
o Translation divided into 3 parts
 initiation
 elongation
 termination
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Transcription
o initiation
o elongation
o termination
Mutations
o substitutions
o deletions
o insertions
DNA inherited by an organism leads to specific traits by dictating the synthesis of
certain proteins. Proteins are the link between genotype and phenotype. This chapter
explores the steps in the flow of information from genes to proteins.
Metabolic defects provided evidence that genes specify proteins
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Archibald Garrod (1909): inferred from "inborn diseases of metabolism"
that genes code for enzymes that catalyze specific reactions in cells.
o E.g. hereditary alkaptonuria (urine turns black upon exposure to air)
caused by lack of enzyme that metabolizes alkapton.
Beadle and Tatum (late 1930's): used auxotrophic mutants of
Neurospora crassa ( a type of fungus) to prove Garrods hypothesis that genes
encode enzymes (i.e. one-gene-one-enzyme hypothesis).
o Wildtype N. crassa normally grows well in minimal medium (salts,
glucose, and biotin). Beadle and Tatum identitified mutants which
could not grow in minimal medium. These mutants grew only if
supplied with all amino acids. N. crassa is a model organism. It is
haploid, so its easy to select for recessive mutants. In diploids, normal
allele masks mutant recessive alleles, so its difficult to select based on
phenotype.
o Auxotroph = nutritional mutant which can not grow on minimal
medium. Grow only if supplied with missing nutrient. (e.g. amino acid
which they can not synthesize on their own).
o Refer to Fig 17.2: Beadle and Tatum mutated N. crassa and then
looked for mutants that differed in their nutritional needs from the
wildtype. They used replica-plating to pinpoint auxotrophic defects
by determining precisely which nutrients had to be added to media to
allow growth. They chose to study Arginine mutants.
Using complementation tests, identified three different
classes of mutants which lacked ability to synthesize the amino
acid arginine. They suspected that each class of mutants lacked
a different enzyme in the metabolic pathway from some
precursor to arginine.
 By plating each class of mutant on minimal media containing a
different intermediate in the metabolic pathway leading to
arginine, they showed that the different classes of mutants
were blocked at a different enzymatic step in the pathway. It
was subsequently shown that each mutant in fact lacked a
particular enzyme in the arg pathway.
 These experiments also showed that the combination of
genetics with biochemistry could be used to work out the steps
in metabollic pathways.
One-gene-one polypeptide hypothesis: further experimentation showed
that genes encode for proteins, not all of which are enzymes. Also, many
proteins have a quaternary structure, and each subunit is encoded by a
different gene.
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Transcription and translation are the two main steps from gene to protein
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Soon after the discovery of the double helix, Francis Crick proposed
that biological information flows from DNA to RNA to protein. This
became known as the central dogma of biology. It was later modified
to incorporate the fact that in some viruses, RNA is copied into DNA
by reverse transcriptases.
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Genes are instructions for making specific proteins. But
proteins are not made from DNA directly. The flow of
information from DNA to protein proceeds through a RNA
intermediate. DNA is first transcribed (copied) into
mRNA which is then translated into protein.
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RNA differs structurally from DNA in 3 ways:
o 1. Has uracyl instead thymine
o 2. Sugar is a ribose not a deoxyribose (RNA has OH at 2' C)
o 3. RNA is single stranded (although it can form Watson-Crick basepairing).
Nucleic acids are linear molecules made from 4 monomers (nucleotides).
Proteins are also polymers, but made from 20 different amino acids.
How does DNA code for protein? Two major steps:
o 1. Transcription: synthesis of complementary RNA from coding
strand of DNA.
 this RNA molecule is called a messenger RNA (mRNA)
 mRNA is synthesized by RNA polymerase.
 mRNA and DNA have same language (i.e. code).
o 2. Translation: synthesis of polypeptide under direction of mRNA.
 involves a change from DNA language into protein language.
 involves mRNA, ribosomes, transfer RNA (tRNA)
The basic mechanisms of transcription and translation are the same for both
eukaryotes and prokaryotes. However, there are some differences (Fig 17.3)
o 1. Prokaryotes lack a nucleus; transcription and translation are
coupled, i.e. ribosomes translate mRNA while transcription is still in
progress. In eukaryotes, mRNA must exit the nucleus to be translated
in cytoplasm.
o 2. Differences in gene structure means that transcripts must be
processed in eukaryotes, but not in prokaryotes.
o
3. In eukaryotes, each gene is transcribed separately. In Prokaryotes,
many genes can be transcribed into a single mRNA (polycistronic
RNA).
The genetic code
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DNA code is a triplet code, i.e. each 3 consecutive bases codes for one
amino acid (Fig 17.4).
64 possible "words" can be made from a triplet code (4x4x4=64). Since there
are 64 possible code words, and only 20 amino acids, some amino acids are
encoded by more than one code word. Thus the genetic code is redundant
(but not ambiguous).
The genetic instructions for a polypeptide chain are written in the DNA as a
series of three-nucleotide words.
For each gene, only one of the two strands is transcribed. This is called the
template or noncoding strand. The other strand functions to make
complementary strand during DNA replication.
mRNA is complementary to DNA template. The mRNA base triplets are called
codons.
During translation, the sequence of codons along a genetic message (mRNA)
is decoded, or translated, into a sequence of amino acids making the
polypeptide chain. Each codon along the mRNA molecule specifies which of
the 20 amino acids will be incorporated at the corresponding position along a
polypeptide. It takes 900 nucleotides along a mRNA strand to make a
polypeptide 300 amino acids long.
Codon Table (Fig 17.5)
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Not all codons code for amino acids
o AUG codes for methionine but also serves as start
codon, i.e. signals start site for translation.
o three codons, (UAA, UAG, UGA) are stop codons,
i.e. signal the end of translation.
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Ability to extract intended message from written language
depends on reading the symbols in the correct sequence of
groupings. This ordering is called the reading frame.
o change in reading frame results in synthesis of
different polypeptide.
o RNA polymerase uses start codon to establish proper
reading frame. All polypeptides have methionine as
first amino acid.
All organisms have the same genetic code, i.e. genetic
code is universal
o most compelling piece of evidence suggesting that all
living things have evolved from a common ancestor.
o Also means that genes from humans can be cloned
into other species and they will specify the same
polypeptide. Useful in biotechnology and genetic
engineering.
Transcription
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Coding stand of DNA is transcribed into mRNA by the enzyme RNA
polymerase (Fig 17.7 and Fig 17.8).
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RNA polymerase: binds promoter region of
genes, pries apart DNA strands, and joins together
RNA nucleotides as they base pair along DNA
template. Adds nucleotides only in the 5' to 3'
direction. Stops transcription when it reaches
sequences at end of gene.
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Transcription occurs in 3 stages:
o 1. RNA binding and initiation of transcription:
 RNA polymerase binds promoter region of gene, recognizes
speficic DNA sequences, pries DNA strands apart and begins
polymerization
 Promoter = DNA sequences upstream of genes responsible for binding
RNA polymerase and transcription factors.
 transcription factors = proteins which bind promoters and RNA
polymerase. Determine when, where, and how much mRNA is produced.
o 2. Elongation of RNA strand:
 RNA polymerase moves along DNA, untwisting helix, separating
the DNA strands (10 bases at a time) and joins RNA nucleotides
together as they base pair to template strand.
 as mRNA is made, it peels away from template
 rate of transcription is about 60 bases/sec.
o 3. Termination of transcription:
 RNA polymerase reaches termination sequence at end of
gene and stops polymerizing.
RNA processing in eukaryotes
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Eukaryotic cells modify their primary transcripts in the nucleus before the
transcripts are dispatched into the cytoplasm to be translated into proteins.
These modifications are referred to as RNA processing (Fig 17.9). The premRNA undergoes 3 modifications:
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Addition of a 5' CAP
o a modified guanine nucleotide is added to 5' end of transcript.
o 5' cap promotes export into cytoplasm, protects mRNA from
degredation, and is necessary for translation.
Addition of a poly-A tail
o An enzyme adds 50-250 adenine nucleotides to 3' end of
transcript, forming poly-A tail.
o Poly-A tail functions in transport of transcript out of nucleus, and
protects mRNA from degredation.
o Poly-A tail is not translated into polypeptide.
Removal of introns
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o
Eukaryotic genes contain noncoding sequences that are not
translated, called introns. The coding sequences that are
o
o
o
translated are called exons (Fig 17.10). The number of introns
and exons varies between different genes.
The removal of introns from primary transcripts is called RNA
splicing, and is carried out by a group of ribonucleoproteins
which assemble to form the spliceosome. Spliceosomes
recognize the intron/exon border sequences, remove the
intervening intron, and then join the exons together, to form a
contiguous coding sequence that is ready for translation (Fig
17.10 - 11).
As a result of RNA splicing, primary transcripts are usually much
longer than mature transcripts.
Alternative splicing can lead to different proteins being
encoded by the same gene. Because of alternative splicing, the
number of different proteins made by an organism is much
larger than the number of genes it encodes.
Translation is RNA-directed synthesis of a polypeptide
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In translation, message in mRNA gets translated (interpreted) into protein.
The interpreter is another type of RNA molecule called transfer RNA (tRNA).
o tRNA = functions to transfer amino acids from the cytoplasm's amino
acid pool to a ribosome. The ribosome adds each amino acid brought
to it by tRNA to the growing end of a polypeptide chain (Fig 17.13)
Structure and function of tRNA (Fig 17.14
o
o
o
o
transcribed from DNA
consists of single RNA strand (~ 80 bases long)
folds back onto itself to form secondary structure
(cloverleaf-like)
3' end of tRNA binds to specific amino acid. Each amino
acid binds to a different tRNA. Each type of tRNA associates with
a particular mRNA codon. This association is based on H-bonding
between codon and complementary bases in anticodon of tRNA.
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A tRNA that binds to an mRNA codon by specifying
a particular amino acid must carry only that amino acid to
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the ribosome.
Each amino acid is matched with the correct tRNA
by a specific enzyme called aminoacyl-tRNA synthase
(Fig 17.15). There's a whole family of these enzymes, one
for each amino acid.
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Ribosomes
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Ribosome holds the tRNA and mRNA molecules close together and catalyzes
the addition of an amino acid to the carboxyl end of a growing polypeptide(Fig
17.16).
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Made of two subunits (large and small). In
eukaryotes, ribosomes are made in
nucleolus.
Each subunit is an aggregate of numerous
proteins associated with another type of
RNA called ribosomal RNA (rRNA).
Has binding site for mRNA and two binding
sites for tRNA, known as P site (peptidyltRNA site) and A site (aminoacyl-tRNA site)
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Translation divided into 3 parts:
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1. Intitiation (Fig 16.17)
Small subunit of ribosome binds mRNA and then binds initiator tRNA at
the first codon. This establisheds the reading frame.
o Large subunit then binds, and exposes the next codon on mRNA at the
A site of ribosome. Ready for elongation.
2. Elongation (Fig 16.18)
o 1. codon recognition: an incoming aminoacyl-tRNA binds to codon at
A-site
o 2. peptide bond formation: peptide bond is formed between new
amino acid and growing polypeptide chain.
o 3. Translocation; tRNA that was in P site is released. tRNA in the A site
is translocated to the P site. In the process, ribosome advances by one
codon.
3 Terminatiuon (Fig 17.19):
o Stop codon reached along mRNA.; ribosome binds a protein called
release factor which hydrolyzes bond between tRNA at P site and last
amino acid of polypeptide. Both tRNA and polypeptide float away.
During and after its synthesis, polypeptide chain begins to coil and fold
spontaneously, to form functional proteins with a specific conformation.
Some polypeptides have signal sequences that target them to specific
destinations in the cell (Fig 17.21)
o
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Mutations
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Mutations are changes inthe genetic makeup of a cell.
Ultimate source of all genetic variation.
Point mutations = changes in just one nucleotide in a single gene. Three
types: substitutions, insertions, and deletions
o Base Pair substitutions (Fig 17.24).
o
o
o
replacement of one nucleotide by another; may or may not
affect amino acid sequence (depends on specific substitution)
silent mutation: new codon still codes for same
amino acid (aka synonomous substitution)(b/c code is
degenerate).
missence mutation; codon changed such that it
encodes a new amino acid ( aka nonsynonomous
substitution) .
o
nonsense mutation: codon changed to stop codon;
causes early chain termination.
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o
o
o
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Base-pair deletion or insertion (Fig 17.25).
addition or loss of a base pair
result in frameshift mutations, alterations of
reading frame. Result in different polypeptides being made,
usually nonfunctional.
Insertion or deletion of 3 nuceotides results in
deletion or insertion of one amino acid, but no frameshift.