One Gene, One Polypeptide
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A gene is defined as a DNA sequence.
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There are many steps between genotype and phenotype; genes cannot by themselves produce a phenotype.
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In the 1940s, Beadle and Tatum showed that when an altered gene resulted in an altered phenotype, that
altered phenotype always showed up as an altered enzyme.
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They experimented with the bread mold Neurospora crassa, an organism whose haploid nuclei and spores
allow for easy detection of recessive mutations.
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Wild-type strains of Neurospora (“original eaters,” or prototrophs) were grown on a minimal nutritional
medium consisting of sucrose, minerals, and a few vitamins.
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Some wild-type Neurospora were then treated with a mutagen, an agent that causes changes in the DNA.
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Some of the treated strains (“increased eaters,” or auxotrophs) could no longer grow on the minimal medium,
but needed additional nutrients.
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These auxotrophs were assumed to have suffered mutations in genes that code for enzymes used to synthesize
the needed nutrients.
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For each auxotrophic strain, Beadle and Tatum were able to find a single compound that could support its
growth.
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For example, the so-called arg mutants could not make their own arginine and needed to be supplemented
with this amino acid from the environment.
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This result suggested that each mutation causes a defect in only one enzyme in a metabolic pathway: the onegene, one-enzyme hypothesis.
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Beadle and Tatum actually found several genetically different arg mutant strains with the same phenotype.
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Mapping studies established that some of these arg mutants had mutations at the same chromosomal locus
and so were different alleles of the same gene; other mutations were at different loci or on different chromosomes, and
were in different genes.
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Beadle and Tatum demonstrated that these different genes participated in the same biochemical pathway—the
pathway leading to arginine synthesis. (See Figure 12.1.)
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If the gene defect affected earlier enzyme steps in the pathway, arginine could still be synthesized from
pathway intermediates. Therefore, these arg mutants could grow on a medium supplemented with these intermediate
substances.
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If the defect was for the enzyme step just before arginine synthesis, the arg mutants could not synthesize
arginine at all and could grow only on a medium supplemented with arginine.
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The gene–enzyme connection has undergone several modifications. It was learned, for example, that some
enzymes are composed of different subunits coded for by separate genes, thus suggesting, instead of the one-gene, oneenzyme hypothesis, a one-gene, one-polypeptide relationship.
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Even this hypothesis has required modification: Some genes code for RNA molecules that are never
translated into polypeptides, and others are involved in controlling which other DNA sequences are expressed.
DNA, RNA, and the Flow of Information
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How is the information encoded in DNA used to specify a particular polypeptide? The expression of a gene
takes place in two steps.
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Transcription makes a single-stranded RNA copy of a segment of the DNA.
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Translation uses information encoded in a portion of the RNA to make a polypeptide.
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In eukaryotes, these two steps are physically separated.
RNA differs from DNA
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RNA consists of only one polynucleotide strand.
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The sugar in RNA is ribose, not deoxyribose.
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Although three of the nitrogenous bases (adenine, guanine, and cytosine) are identical, the fourth base in
DNA is thymine, whereas in RNA it is uracil (similar to thymine but lacking the methyl group).
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RNA can base-pair with single-stranded DNA (with adenine pairing with uracil instead of thymine as in
complementary base-pairing of DNA) and also can fold over and base-pair with itself.
Information flows in one direction when genes are expressed
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Francis Crick’s central dogma stated that DNA codes for RNA, and RNA codes for protein; that is, once
information passes into protein, it cannot get out again. (See Figure 12.2.)
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Messenger RNA, or mRNA (a complementary copy, formed via transcription of one DNA strand of a
particular gene ) moves from the nucleus of eukaryotic cells into the cytoplasm, where it serves as a template for
protein synthesis.
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Transfer RNA, or tRNA, is the link (via the process of translation) between the code of the mRNA and the
amino acids of the polypeptide, thus specifying the correct amino acid sequence in a protein. (See Figure 12.3.)
RNA viruses modify the central dogma
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Certain viruses (e.g., influenza, poliovirus) use RNA rather than DNA as their information molecule during
transmission.
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These viruses transcribe from RNA to RNA; they make an RNA strand that is complementary to their
genome and then use this “opposite” strand to make multiple copies of the viral genome by transcription.
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HIV and certain tumor viruses (called retroviruses) have RNA as their infectious information molecule; they
convert it to a DNA copy inside the host cell and then use it to make more RNA.
Transcription: DNA-Directed RNA Synthesis
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In normal prokaryotic and eukaryotic cells, transcription (formation of a specific RNA from a specific DNA)
requires the following:
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A DNA template for complementary base pairing.
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The appropriate ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) to act as substrates.
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The enzyme RNA polymerase.
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Just one DNA strand (the template strand) is used to make the RNA; the complementary non-template strand
remains untranscribed.
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For different genes in the same DNA molecule, however, the roles of these strands may be reversed.
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The continuous double helix of DNA has many regions that are read by RNA polymerase.
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The DNA double helix partly unwinds to serve as template.
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As the RNA transcript forms, it peels away, allowing the already-transcribed DNA to be rewound into the
double helix.
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(See Animated Tutorial 12.1.)
Initiation of transcription requires a promoter and an RNA polymerase
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The first step of transcription, initiation, begins at a promoter, which is a special sequence of DNA to which
RNA polymerase binds very tightly.
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There is at least one promoter for each gene (or sets of genes in prokaryotes) to be transcribed into mRNA.
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The RNA polymerase binds to the promoter region when conditions allow.
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The promoter sequence directs the RNA polymerase as to which of the double strands is the template and in
what direction the RNA polymerase should move, serving, in effect, as punctuation marks for the transcription process.
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RNA is synthesized in the 5-to-3 direction, moving along the template DNA in the 3-to-5 direction. (See
Figure 12.4.)
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Not all promoters are identical. Some bind RNA polymerase more effectively; this causes them to be
transcribed more frequently when other conditions allow.
RNA polymerase elongates the transcript
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After binding, RNA polymerase unwinds the DNA about 20 base pairs at a time and reads the template in the
3-to-5 direction (elongation). (See Figure 12.4.)
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The new RNA elongates from its 5 end to its 3 end; thus the RNA transcript is antiparallel to the DNA
template strand.
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Transcription errors for RNA polymerases are high relative to DNA polymerases (with a mistake occuring for
every 104 to 105 bases incorporated). These are errors in the copies, however, not in the original DNA master, so they
are less likely to be as harmful as mutations in DNA.
Transcription terminates at particular base sequences
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Particular base sequences in the DNA specify termination.
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Gene mechanisms for termination vary.
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For some genes, the newly formed transcript simply falls away from the DNA template and the RNA
polymerase.
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For other genes, a helper protein pulls the transcript away.
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In prokaryotes, translation of the mRNA often begins before transcription is complete.
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In eukaryotes, the process is more complicated and involves a spatial separation as well as further processing.
The Genetic Code
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A genetic code relates genes (DNA) to mRNA and mRNA to the amino acids of proteins, specifying which
amino acids will be used to build a protein by the transcription process.
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mRNA is read in three-base contiguous segments called codons.
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The number of different codons possible is 64 (43), because each position in the codon can be occupied by
one of four different bases.
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The 64 possible codons code for only 20 amino acids and the start and stop signals found in all mRNA
molecules.
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AUG, which codes for methionine, is called the start codon, the initiation signal for translation.
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Three codons (UAA, UAG, and UGA) are stop codons, which direct the ribosomes to end translation.
The genetic code is redundant but not ambiguous
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acids.
After subtracting start and stop codons, the remaining 60 codons code for 19 different amino acids.
This means that many amino acids have more than one codon. Thus, the code is redundant.
But the code is not ambiguous. Each codon is assigned only one amino acid, not two or three possible amino
The genetic code is (nearly) universal
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The genetic code is nearly universal, applying to all species on our planet. (See Figure 12.5.)
Minor variations are found within mitochondria and chloroplasts; other exceptions are few and slight.
This common genetic code has great implications in genetic engineering.
Biologists deciphered the genetic code by using artificial messengers
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In the early 1960s, molecular biologists broke the genetic code determining how only four bases (A, U, G,
and C) could code for 20 different amino acids.
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The existence of a three-letter codon was postulated early on because only a triplet code could contain up to
64 (4  4  4) codons.
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Nirenberg prepared an artificial mRNA in which all bases were uracil (poly U).
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When incubated with required additional components, the poly U mRNA led to synthesis of a polypeptide
chain consisting only of phenylalanine amino acids.
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UUU appeared to be the codon for phenylalanine.
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Other codons were deciphered from this starting point.
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An additional technique finished the deciphering.
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Simple synthetic mRNAs only three nucleotides long could bind to ribosomes.
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This complex then caused the tRNA-amino acid to bind according to the three-letter codon.
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Using this technique, the code was fully deciphered. (See Figure 12.6 and Animated Tutorial 12.2.)
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Radioactive labeling was also used to decipher the code.
Preparation for Translation: Linking RNAs, Amino Acids, and
Ribosomes
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The molecule tRNA is required to assure specificity in the translation of mRNA into proteins.
The tRNAs must read mRNA correctly.
They also must carry the correct amino acids.
(See Video 12.1.)
Transfer RNAs carry specific amino acids and bind to specific codons
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The codon in mRNA and the amino acid in a protein are related by way of an adapter—a specific tRNA
molecule that carries (is “charged with”) an amino acid, associates with mRNA molecules, and interacts with
ribosomes.
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A tRNA molecule has 75 to 80 nucleotides and a three-dimensional shape (conformation) maintained by
complementary base pairing and hydrogen bonding. (See Figure 12.7.)
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The three-dimensional shape of the tRNAs allows them to combine with the binding sites of the ribosome.
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At the 3 end of every tRNA molecule is a site to which its specific amino acid binds covalently.
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Midpoint in the sequence are three bases called the anticodon.
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The anticodon is the contact point between the tRNA and the mRNA.
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The anticodon is complementary (and antiparallel) to the mRNA codon.
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The codon and anticodon unite by complementary base pairing.
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There are fewer anticodon codes than mRNA codons because some codon–anticodon interactions tolerate a
mismatch at the 3 base of the mRNA, a phenomenon called wobble. (See Figure 12.5.)
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Wobble is allowed in some matches but not in others. It does not allow the genetic code to be ambiguous.
Activating enzymes link the right tRNAs and amino acids
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The correct amino acids are attached to the correct tRNAs by a family of activating enzymes called
aminoacyl-tRNA synthetases.
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Each activating enzyme is specific for one amino acid and its tRNA.
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The enzyme has a three-part active site that binds a specific amino acid, ATP, and a specific tRNA, which is
charged with a high-energy bond.
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This bond provides the energy for making the peptide bond that will join adjacent amino acids.
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The reactions have two steps (see Figure 12.8):
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Enzyme + ATP + AA  enzyme—AMP—AA + PPi
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Enzyme—AMP—AA + tRNA  enzyme + AMP + tRNA—AA
The ribosome is the workbench for translation
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Each ribosome has two subunits, a larger one and a smaller one. (See Figure 12.9.)
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In eukaryotes the large one has three different associated rRNA molecules and 45 different proteins.
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The smaller subunit has one rRNA and 33 different protein molecules.
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When they are not translating, the two subunits are separate.
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Ribosomes of prokaryotes are somewhat smaller, and their ribosomal proteins and rRNAs are different.
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The different proteins and rRNAs are held together by ionic bonds and hydrophobic forces, not covalent
bonds.
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The structure can self-assemble if disassembled by detergents.
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Ribosomes are simply molecular factories and are nonspecific. They combine with any mRNA and all
tRNAs.
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The large subunit has four sites where tRNA molecules bind. (See Figure 12.9.)
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The T site is where the tRNA first lands. It is brought to the site by a special protein, the T, or transfer, factor.
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The A site is where the tRNA anticodon binds to the mRNA codon.
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The P site is where the tRNA adds its amino acid to the growing polypeptide chain.
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The E (exit) site is where the tRNA, less its amino acid, goes before leaving the ribosome.
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The small ribosomal subunit plays a role in validating the three-base-pair match between the mRNA and the
tRNA. If hydrogen bonds have not formed between all three base pairs, the tRNA is ejected from the ribosome.
Translation: RNA-Directed Polypeptide Synthesis
Translation begins with an initiation complex
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At the beginning of translation, an initiation complex forms consisting of a charged tRNA bearing its amino
acid and a small subunit of the ribosome, both bound to the mRNA. (See Figure 12.10.)
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This complex is bound to a region upstream (toward the 5 end) of where the actual reading of the mRNA
begins.
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The start codon (AUG) for methionine designates the first amino acid in all proteins. (However, some
proteins are trimmed after synthesis, and the methionine is thereby removed.)
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The large subunit then joins the complex.
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The process is directed by proteins called initiation factors.
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(See Animated Tutorial 12.3.)
The polypeptide elongates from the N terminus
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site.
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Ribosomes move in the 5-to-3 direction on the mRNA. (See Figure 12.11.)
They synthesize the peptide in the N terminus-to-C terminus direction.
The large subunit catalyzes two reactions:
Breakage of the bond between the tRNA in the P site and its amino acid (on the polypeptide).
Peptide bond formation between this (tRNA-attached) amino acid and the one attached to the tRNA in the A
This is called peptidyl transferase activity.
rRNA acts as the catalyst for this reaction.
Elongation continues and the polypeptide grows
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After the first tRNA releases methionine, it dissociates from the ribosome and returns to the cytosol.
The second tRNA, now bearing a dipeptide, then moves to the P site.
The next charged tRNA enters the open A site.
The peptide chain is then transferred to the P site.
These steps are assisted by proteins called elongation factors.
A release factor terminates translation
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When a stop codon—UAA, UAG, or UGA—enters the A site, a release factor and a water molecule enter the
A site, instead of an amino acid. (See Figure 12.12.)
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The newly completed protein then separates from the ribosome.
Regulation of Translation
Some antibiotics and bacterial toxins work by inhibiting translation
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Antibiotics are defensive molecules produced by some fungi and bacteria and which often destroy other
microbes.
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They have been used since the 1940s to combat human bacterial infectious disease.
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Antibiotics must specifically destroy microbial invaders but not harm the human host.
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Some antibiotics work by blocking the synthesis of the bacterial cell walls, others by inhibiting protein
synthesis. (See Table 12.2.)
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Because of differences between prokaryotic and eukaryotic ribosomes, the human ribosomes are unaffected.
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Some bacteria affect their human hosts through mechanisms similar to those we use against them. In the
disease diphtheria (against which children now are vaccinated), the infective agent Cornybacterium diphtheriae,
produces a highly lethal toxin that modifies and inactivates a protein that is essential for the movement of mRNA and
ribosomes during eukaryotic protein synthesis.
Polysome formation increases the rate of protein synthesis
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Polysomes are mRNA molecules with more than one ribosome attached. (See Figure 12.13.)
These make protein more rapidly, producing multiple copies of protein simultaneously.
Posttranslational Events
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Some proteins require additional modification after synthesis before they become functional.
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New chemical groups might be added to the protein, it might be folded (with the assistance of other proteins),
or it might get trimmed.
Chemical signals in proteins direct them to their cellular destinations
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See Figure 12.14 for an illustration of the possible destinations for a newly translated protein.
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As the polypeptide chain forms, it spontaneously folds into its three-dimensional shape.
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The amino acid sequence also contains an “address label” indicating where in the cell the polypeptide
belongs.
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All protein synthesis begins on free ribosomes in the cytoplasm.
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In eukaryotes, as the peptide chain is made, information on the nascent portion gives one of two sets of
instructions:
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Finish translation and be released to the cytoplasm.
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Stall translation, go to the endoplasmic reticulum, and finish synthesis at the ER surface.
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Those polypeptides destined to finish synthesis in the cytoplasm may contain information in their amino acid
sequence that specifies where they belong: the nucleus, mitochondria, or peroxisomes.
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Some of the proteins that are transported to a destination require chaperonin proteins and receptor docking
proteins.
• The protein binds to the docking protein at the outer membrane of the appropriate organelle.
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A channel opens in the membrane, allowing the protein to pass through.
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In the process, the protein usually is unfolded by a chaperonin so that it can pass through the channel, after
which it refolds to its normal conformation.
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Those destined for the ER generate an approximately 25-amino-acid-long hydrophobic leader sequence that
signals to a signal recognition particle, which is composed of protein and RNA. (See Figure 12.15.)
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The association of the signal to the signal receptor particle stalls any additional translation.
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This stalling continues until the ribosome attaches to a specific receptor protein on the surface of the ER.
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Translation continues with the protein moving through a pore in the ER membrane.
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Some proteins have signals that direct the embedding of the protein into the ER membrane.
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This is when membrane proteins of the ER, Golgi apparatus, lysosomes, and plasma membrane get
positioned.
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Other signals direct the protein to the Golgi apparatus, lysosomes, or to the outside of the cell.
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Proteins with no signals from the ER go through the Golgi apparatus and are secreted from the cell.
Many proteins are modified after translation
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It is the exception, not the rule, that the finished protein is identical to the translation from the mRNA code.
Modifications are often essential to the final functioning of the protein. (See Figure 12.16.)
Proteolysis is the cleavage of the protein to make a shortened finished protein.
Insulin is an example of a protein that gets trimmed.
The signal to go to the ER is often cleaved after the protein gets there.
The virus HIV needs a protease to cleave a protein. One treatment for HIV inhibits this enzyme.
Glycosylation involves the addition of sugars to the protein.
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Signals in the amino acid sequence of the protein direct the addition of the sugars in the ER by resident
enzymes.
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Additional modifications occur in both the Golgi apparatus and the ER.
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Phosphorylation is the addition of phosphate groups to certain proteins. These additions may be temporary
and affect the activity of the protein by changing its three-dimensional shape.
Mutations: Heritable Changes in Genes
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Mutations are heritable changes in DNA—changes that are passed on to daughter cells.
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In single-celled organisms, any mutations that occur are passed to the daughter cells at the time of cell
division.
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Multicellular organisms have two types of mutations:
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Somatic mutations are passed on during mitosis, but the affected cells never become gametes and so do not
pass to subsequent generations.
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Germ line mutations are mutations that occur in cells that might give rise to gametes.
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Some mutations cause visible phenotypic change. Others cause metabolic changes that may not be readily
detectable.
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Some mutations, called conditional mutants, exert their effect only under certain restrictive conditions.
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They are unaffected under permissive conditions, but express the mutant phenotype at the restrictive
condition.
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A temperature-sensitive mutant allele, for example, may code for an enzyme that is altered at the restrictive
temperature.
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All mutations are alterations of the DNA nucleotide sequence and are of two types:
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Point mutations are mutations of single genes.
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Chromosomal mutations are changes in the arrangements of chromosomal DNA segments.
Point mutations are changes in single nucleotides
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Point mutations result from the addition or subtraction of a nucleotide base or the substitution of one base for
another.
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Point mutations can occur as a result of mistakes during DNA replication or can be caused by environmental
mutagens, such as chemicals and radiation.
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Because of redundancy in the genetic code, some point mutations, called silent mutations, result in no change
in the amino acids in the protein.
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Some mutations, called missense mutations, cause an amino acid substitution.
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An example in humans is sickle-cell anemia, a defect in the -globin subunits of hemoglobin.
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The red blood cells collapse when oxygen levels are low. (See Figure 12.17.)
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Missense mutations may reduce the functioning of a protein or disable it completely.
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Nonsense mutations are base substitutions that cause a change from a codon that instructs the incorporation
of an amino acid to a codon that terminates translation.
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A frame-shift mutation consists of the insertion or deletion of a single base in a gene.
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This causes the most disruption when the event occurs at or near the beginning of the template.
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This type of mutation shifts the code, changing many of the codons to different codons.
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These shifts almost always lead to the production of nonfunctional proteins.
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(See Video 12.2.)
Chromosomal mutations are extensive changes in the genetic material
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DNA molecules can break and re-form.
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This can cause four different types of mutations: deletions, duplications, inversions, and translocations. (See
Figure 12.18.)
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Deletions are a loss of a chromosomal segment.
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Duplications are a repeat of a segment.
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Breaking and rejoining leads to inversions if segments get reattached in the opposite orientation.
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Translocations result when a portion of one chromosome attaches to another.
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Translocations can be reciprocal (see Figure 12.18d) or nonreciprocal.
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Translocations often lead to duplications and deletions, and may lead to sterility if normal chromosome
pairing in meiosis cannot occur.
Mutations can be spontaneous or induced
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Spontaneous mutations are permanent changes that occur without outside influence. (See Figure 12.19.)
Spontaneous mutations may be caused by any of several mechanisms.
Nucleotides occasionally change their structure (called a tautomeric shift).
A base may temporarily change to its unusual tautomer at the same time that replication is occurring.
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The tautomer may pair with the alternate purine if it is a purine, or the alternate pyrimidine if it is a
pyrimidine.
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DNA polymerase sometimes makes errors in replication.
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These errors are often repaired by the proofreading function of the replication complex, but some errors
escape and become permanent.
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Meiosis is imperfect. Nondisjunction can occur. Random chromosome breaks rejoin incorrectly, leading to
translocations.
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Induced mutations are permanent changes caused by some outside agent.
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Some chemicals alter covalent bonds in nucleotides.
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Nitrous acid deaminates cytosine, converting it to uracil.
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DNA polymerase mistakes uracil for thymine and inserts an A during replication instead of a G.
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Benzoapyrene, a product of incomplete combustion that is found in all smoke, adds a large chemical group to
guanine, making it unavailable for base pairing. Any base may then be inserted to fill the gap, and of course, threefourths of the time it is the wrong base.
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Radiation damages DNA.
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Ionizing radiation (X rays) produces highly reactive compounds and atoms called free radicals.
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Gamma rays also produce free radicals.
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Free radicals can alter bases or break the sugar–phosphate backbone, causing chromosomal abnormalities.
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Ultraviolet radiation is absorbed by pyrimidines in the DNA, and when two thymines or two cytosines are
next to each other on the same strand of a double-stranded DNA molecule, a covalent bond can form.
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Their interstrand covalent bonds make the DNA unreplicable.
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The long-term benefit of mutations is that they provide genetic diversity for evolution to work on.
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The possible detrimental effects of mutation are the outright death of the organism or a poor fit between the
organism and its environment.
Mutations are the raw material of evolution
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Mutations are rare events and most of them are point mutations involving one nucleotide.
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Frequency of mutations is much lower than one mutation per 10 4 genes per DNA duplication. Sometimes
they are as rare as one per 109 genes per duplication.
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Different organisms vary in mutation frequency.
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Mutations can be detrimental, neutral, or occasionally beneficial.
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Humans have 20 times the genes of a prokaryote, due partially to duplication of DNA sequences and then to
divergence of the sequences over time.
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Random accumulation of mutations in the extra copies of genes can lead to the production of new useful
proteins.