Download Chapters 16-17 (DNA and protein synthesis)

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Chapter 16: the Molecular Basis of Inheritance
Important Scientists
Beadle and Tatum: used bread mole and discovered the “one gene-one enzyme” hypothesis (genes are
responsible for the production of enzymes)
Hershey and Chase: exposed E.coli to viruses and discovered that the DNA, not the protein, is the carrier
of genetic information
James Watson and Francis Crick: discovered the structure of DNA
Maurice Wilkins and Rosalind Franklin: did an X-ray crystallography of DNA
Erwin Chargaff: the amounts of complement bases are equally (A=T; C=G)
Meselson and Stahl: discovered semi-conservative replication
Nucleic Acids
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Nucleic acids store and transmit genetic information
- DNA is the genetic material that is inherited from one generation to the next and is
reproduced in each cell of an organism
- The instructions in DNA are “copied” to RNA, ribonucleic acid, which directs the synthesis of
proteins
- The sequence of nucleotides in DNA ultimately determines the sequence of amino acids in
protein
A nucleic acid is a polymer of nucleotides
A nucleotide is composed of three parts:
1. Pentose (5 carbon) sugar
-either ribose (RNA) or deoxyribose (DNA)s
2. A nitrogenous base
-there are 4: adenine, thymine, cytosine, guanine (uracil for RNA)
-can be purines (double ring) or pyridimines (single-ring)
3. A phosphate group
DNA Structure
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DNA molecules consist of two strands called a double helix
“Sugar-phosphate backbone” of DNA= the 2 sides of the DNA molecules that are made of
sugars and phosphates
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The “rungs” of the DNA molecule are made of 2 nitrogenous bases
- Cytosine --- Guanine
- Thymine --- Adenine
- Held together by weak hydrogen bonds
- Purines are paired with pyrimidines
A – – – – – – – T
G – – – – – – – C
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 Antiparallel structure:
The sugar-phosphate backbones are facing opposite directions
- Because the strands are parallel but run in opposite directions, the structure is called
antiparallel
- The 2 different directions are 5’ to 3’ and 3’ to 5’
(5’ means that there is a phosphate on carbon 5)
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Thus, a strand of DNA has polarity, with a 5’ end where the 1st nucleotide’s phosphate group
is exposed, and the other 3’ end where the nucleotide has a hydroxyl group on its 3’ carbon
Bonding
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Phosphodiester bonds = covalent bonds between sugars and phosphates and between
sugars and bases
Hydrogen bonds—between base pairs
Hydrogen bonds are weaker than phosphodiester bonds, so they can be broken easier
DNA structure:
sugars (deoxyribose)
phosphate group
Nitrogenous bases
adenine = thymine
30%
30%
guanine
= cytosine
Erwin Chargaff’s
rules:
A=T
20%
20%
C=G
- Each complement base is equal in amount
- All four together make 100%
- For example, if adenine is 35%, what are the rest of the bases?
A = 35%
T = 35%
C = 15%
G = 15%
DNA Synthesis
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Occurs during the S phase of the cell cycle, when chromosomes are long and skinny.
DNA, not proteins, is the carrier of genetic information. (Hershey and Chase)
Each side of the DNA helix is an exact complement to the other. When the 2 sides of a DNA
molecule separate, each strand serves as a template for replication.
Getting Started:
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Binding of Enzymes to Existing DNA:
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Helicase- unwinds double helix
Topoisomerase- prevents tangling of helix and relieves strain of the twisting of DNA
Single strand binding proteins- prevent strands from rejoining
RNA primase- prepares DNA for replication by putting down 3-5 RNA nucleotides to
which the DNA nucleotides can attach
*DNA polymerase can only attach to previously existing DNA strands
 DNA polymerase I
- breaks hydrogen bonds between nitrogenous bases
- brings in new DNA nucleotides to replace RNA nucleotides
- works 5’ to 3’
 DNA polymerase III- proofreads” for errors
A replisome consists of all of the enzymes plus the strand of DNA being copied. A replisome
moves in both directions.
Replication begins at replication origins: where proteins that initiate replication bind to a
specific sequence of nucleotides and separate the 2 strands to form a replication “bubble”
 Multicellular organisms: have many replication origins and replisome
- DNA “unzips” at many places
- This is more efficient  replication is faster
 Bacteria: have one replication origin and replisome
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- Bacteria have a single loop chromosome
Replication proceeds in both directions in the two Y-shaped replication forks
- The replication origin becomes the replication fork.
The “unzipping” results in exposed bases on each side of the DNA ladder
The enzyme RNA primase first adds a short strand of RNA nucleotides, called a RNA primer, to
being the replication process.
- This is because DNA polymerase can only add new nucleotides to a pre-existing strand of
DNA
- The RNA nucleotides will later be replaced with DNA nucleotides
Synthesizing a New DNA Strand
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DNA polymerase begins adding new DNA nucleotides and connecting nucleotides to the
growing end of a new DNA strand, once a few nucleotides of RNA primer are in place.
- Remember: a nucleotide consists of sugar (deoxyribose), a phosphate group, and a
nitrogenous base (A, T, G, or C)
The nitrogenous base sequence of the existing DNA strand determines the base sequence of the
complement strand
- A nucleotide lines up with its complementary base on the template strand.
- It loses 2 phosphate groups and the hydrolysis of this pyrophosphate to 2 inorganic
phosphates provides the energy for polymerization
DNA polymerase III and I are involved in replication of E. coli
- At least 11 different DNA polymerases have been discovered so far in eukaryotes
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Antiparallel Elongation
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The synthesis of the two new sides of the DNA molecule occurs in opposite directions
due to the antiparallel structure of DNA
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Remember: the 2 strands of DNA are antiparallel which means their sugar-phosphate
backbones run in opposite directions
**Replication always goes from 5’ to 3’ because DNA polymerase adds
nucleotides only to the free 3’ end of a growing DNA strand.
The leading strand is the new continuous strand that is synthesized continuously into the
replication fork.
- This is called continuous replication
- A “sliding clamp” protein moves DNA polymerase II along the template strand in the
progressing replication fork
The lagging strand is the new strand that is synthesized in short segments away from the
replication fork
- This is called discontinuous replication
- These short pieces are called Okasaki fragments and are eventually joined together by an
enzyme called DNA ligase
The overall process results in two identical copies of the DNA molecule
Semiconservative replication: the 2 new daughter DNA molecules have one parental strand and one
newly formed strand. Each DNA molecule consists of half “old” and half “new” DNA
DNA replication at the replication fork:
 The DNA molecule unzips
 DNA polymerase adds DNA nucleotides from 5’ to 3’
 DNA polymerase adds new DNA nucleotides
continuously INTO the replication fork on the leading
strand
 DNA polymerase adds new DNA nucleotides OUT of
the replication fork on the lagging strand
 The lagging strand is added in pieces called Okazaki
fragments
 DNA ligase is an enzyme that connects the fragments
of the lagging strand
 The parental strands are the template or mold
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-DNA unzips at many replication origins, which is
more efficient so replication is faster
-The replication bubbles expand
-The red strands = the new strands being added by
DNA polymerase
-the blue = the parental strands
Semiconservative replication:
A ---T
G ---C
T ---A
C ---G
The parental strand
is the template
A
T
A
T
A
T
G
C
G
C
G
C
T
A
T
A
T
A
The
C enzyme helicase
G
unzips the DNA
molecule
-DNA polymerase
C
G adds
C new
G DNA nucleotides
-two identical strands of DNA: ½ is the parental
strand, ½ is newly formed
Proofreading and Repairing DNA
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Initial pairing errors in nucleotide placement occurs as often as 1 per 100,000 base pairs
- However, the amazing accuracy of DNA replication is actually 1 error in 10 billion
nucleotides
DNA polymerases check each newly added nucleotide against its template and remove
incorrect nucleotides.
- The likelihood of mistakes occurring is reduced because the enzyme DNA polymerase
proofreads and corrects any errors that occur during replication
Other enzymes also fix incorrectly paired nucleotides, called mismatch repair.
- Most mutations are known as mismatches because they consist of base pairs that cannot
form hydrogen bonds (like adenine and cytosine- they can never pair up)
Reactive chemicals, radioactive missions, X-rays, and UV light may alter DNA molecules.
- Mutagens are chemicals that are environmental factors that cause mutations
- Mutations that persist to the next cell division are inherited by the daughter cells
- Many scientists believe that the accumulation of many of these mutations over a lifetime
may result in different types of cancer
A large number of different types of DNA repair enzymes correct these
` changes
In nucleotide excision repair, the mutations are cut out by a nuclease.
DNA polymerase correctly fills in the correct nucleotides
DNA ligase connects the new fragment
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Replicating the Ends of DNA molecules
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Because DNA polymerase cannot attach nucleotides to the 5’ end of a daughter DNA strand,
repeated replications cause a progressive shortening of linear DNA molecules.
Telomeres are the ends of chromosomes.
The enzyme telomerase postpones the erosion of genes and prevents the shortening of
chromosomes over time. It ensures that chromosomes stay intact and are correctly replicated.
The shortening of telomeres may limit cell division.
In eukaryotic germ cells, however, the enzyme telomerase lengthens telomeres.
Some somatic cancer cells and “immortal” strains of cultured cells produce telomerase and are
thus capable of unregulated cell division.
DNA and Chromosomes
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The double stranded, circular DNA molecule of a bacterial chromosome is supercoiled and
tightly packed into a region of cell called the nucleoid.
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In eukaryotes, each chromosome exists as chromatin: extremely long DNA double helix
wrapped around a large amount of histone proteins.
- Histones are small positively-charged proteins that bind to the negatively charged DNA.
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DNA is wrapped around histone proteins, and histones bundle to make nucleosomes.
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Nucleosomes are the basic units of DNA packing.
Nucleosomes consist of 8-10 histones
Unfolded chromatin looks like a string of beads, each bead a nucleosome consisting of the
DNA helix wound around a protein core of four pairs of different histones.
In interphase, chromatin is extended in the nucleus, whereas condensed chromosomes appear
during mitosis.
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Chapter 17: From Gene to Protein
Protein Synthesis
DNA
protein
(nucleus)
(ribosome)
1. Transcription
DNA
mRNA
-in the nucleus
-uses the same “language” of nucleic acids
*mRNA processing
-“splicing”
-in the nucleus
2. Translation
mRNA
amino acids
(tRNA and
rRNA
are also
involved)
polypeptide (primary structure)
protein
-changes the “language” of nucleotides to amino acids
-takes place on ribosomes = sites of translation and polypeptide synthesis
DNA vs. RNA
DNA
RNA
-purpose: produces the chromosomes in
daughter cells
-double helix
-when: S-phase of cell cycle
Both use
-polymer of nucleotides
DNA as a
-deoxyribose (sugar)
template
-phosphate group
-4 nitrogen bases:
-A, C, G, T
-All of the DNA molecule is replicated, so
daughter strands are identical
-purpose: uses instructions in genes of DNA to
produce a protein (=polypeptide)
-single strand
-when: throughout cell cycle
-polymer of nucleotides
-ribose (sugar)
-phosphate group
-4 nitrogen bases:
-A, C, G, U (uracil)
-Only a section of DNA (=a gene) is replicated to
code for a protein
Genetic Material
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Gene expression is the DNA-directed synthesis of proteins (or RNAs)
RNA is the link between a gene and the protein for which it codes.
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Genetic code
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Genetic material consists of two types of biomolecules: DNA and RNA
- Both are nucleic acids and both store genetic information
- Nucleic acids consist of a long strand of repeating subunits that act as letters in a code
- Base sequence and base pairing provide the basis of the genetic code
Codons: Triplets of Bases
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The translation of nucleotides into amino acids uses a triplet code, or codon, to specify each
amino acid.
- There are 64 different codons, which each code for one of the 20 amino acids
The base triples along the template strand of a gene of DNA are transcribed into mRNA codons.
The same strand of a DNA molecule can be the template strand for one gene and the
complementary strand for another.
The mRNA is complementary to the DNA template since its bases follow the same base-pairing
rules, with the exception that uracil substitutes for thymine in RNA.
The mRNA base triplets are called codons,
*The codons are read in the 5’  3’ direction
The codon AUG is the “start codon” and codes for methionine. There are 3 possible stop
codons: UAA, UGA, UAG.
- The start codon is an amino acid, but the stop codons are not.
A tRNA molecule carries a triplet called an anti codon which is the complement to the codon
and carries the correct amino acid
The code is often redundant, meaning that more than one codon may specify a single amino
acid. But no codon specifies two different amino acids.
The nucleotide sequence on mRNA is read in the correct reading frame, starting at a start codon
and reading each triplet sequentially.
Evolution of the Genetic Code
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The genetic code of codons and their corresponding amino acids is almost universal.
- A bacterial cell can translate the genetic messages of human cells.
The near universality of a common genetic language provides compelling evidence for the
evolutionary connection of all living organisms.
3 types of RNA
mRNA or messenger RNA
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Function: carries copies of the instructions for assembling amino acids from DNA to the rest of
the cell. It is a temporary copy of a gene that encodes a protein
Converted from DNA through the process of transcription
rRNA or ribosomal RNA
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Function: combines with proteins to make up ribosomes
80% of the RNA in a cell is rRNA
tRNA or transfer RNA
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Function: transfers each amino acid to the ribosome to help assemble protein.
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Protein Synthesis
Transcription
How DNA is converted to RNA
gene
A ---T
G ---C
T ---A
C ---G
goes back
to DNA
A
G
T
C
U
C
A
G
T
C
A
G
nucleus
mRNA leaves through
nuclear pores
Molecular Components of Transcription
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DNA never leaves the nucleus, so it’s converted into mRNA which can leave the nucleus
- RNA is like a disposable copy of a DNA segment
- RNA polymerase
-unwinds the DNA double helix
-breaks hydrogen bonds between nitrogen bases in DNA
-adds new RNA nucleotides
-binds only to DNA promoters, which have specific base sequences.
The promoter is the DNA sequence where RNA polymerase attaches and initiates transcription.
In bacteria, the terminator is the sequence that signals the end of transcription.
A transcription unit is the sequence of DNA that is transcribed into one RNA molecule.
Eukaryotes have 3 RNA polymerases in the nuclei and each is responsible for making the 3 types
of RNA. Prokaryotes have one type of RNA polymerase.
Only one strand of the DNA (=the template/sense/master strand) directs the synthesis of RNA
Initiation of Transcription
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The enzyme RNA polymerase attaches to a specific region of DNA
The promoter is the binding site for RNA polymerase and determines where transcription starts
and which DNA strand is used as the template.
- It includes recognition sequences such as the TATA box common in eukaryotes, upstream
from the start point.
In eukaryotes, transcription factors must first recognize and bind to the promoter before RNA
polymerase II can attach.
- This unit is called the transcription initiation complex.
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Elongation of the RNA Strand
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RNA polymerase untwists and unwinds the DNA, exposing the DNA nucleotides for base pairing
with RNA nucleotides
- RNA polymerase joins the nucleotides to the 3’ end of the growing polymer.
It moves along the DNA away from the promoter site as it builds a single complementary strand
of RNA, called a primary transcript.
- The new RNA peels away from the DNA template and the DNA double helix re-forms.
The sequence of DNA nucleotides determines the sequence of RNA chain.
Several molecules of RNA polymerase may be transcribing simultaneously along a gene, allowing
a cell to be more efficient and produce more mRNA.
Termination of Transcription
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RNA polymerase reaches the terminator region, or the end of the DNA to be transcribed
- *RNA polymerase and the primary transcript are released from the DNA
-Proteins cut the pre-mRNA loose.
-RNA polymerase eventually falls off the DNA after transcribing hundreds more
nucleotides.
In prokaryotes, transcription ends after RNA polymerase transcribes the terminator sequence.
In eukaryotes, polymerase continues past a polyadenylation signal sequence (AAUAAA).
mRNA processing
1. “Splicing” = introns are removed and exons are joined by ligase
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Introns = intervening
Exons = expressed
2. A methyl guanine cap is added to the beginning of mRNA
3. A poly A tail (10-25 adenines) is attached to the end
- The longer the poly-A tail, the longer the life span of a particular mRNA
- It also helps transport mRNA out of the nucleus and helps the mRNA attach to a ribosome
and begin translation
 This process takes place in the nucleus
 The “cap and tail” provides protection against enzymes that break down nucleic acids
 Why is this process important?
- Only certain information is needed for specific jobs to create different sequences, and
sometimes there is more information than needed in the mRNA strand.
- Processing mRNA before it’s translated makes translation faster and more efficient because
there are less codons to be changed into amino acids.
- If introns are left in RNA, the consequences can be serious
mRNA splicing
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Some introns are involved in regulating gene activity, and splicing is necessary for the export of
mRNA from the nucleus.
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Alternative RNA splicing allows some genes to produce different polypeptides.
Exons may code for polypeptide domains, which are the functional parts of a protein (such as
binding and active sites).
Translation
Molecular Components of Translation
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Transfer RNA (tRNA) molecules carry specific amino acids to ribosomes.
They each have a base triplet, called an anticodon, that base-pairs with a complementary codon
on mRNA.
- Allows amino acids to be arranged in the sequence prescribed by transcription from DNA.
The Structure and Function of Transfer RNA (tRNA)
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tRNA is transcribed in the nucleus of a eukaryote and moves into the cytoplasm where it can be
used repeatedly.
These single-stranded, short RNA molecules are arranged into a cloverleaf shape by hydrogen
bonding between complementary base sequences and fold into a 3-D L-shaped structure.
- The anti codon is at one end of the L; the 3’ end is the attachment site for its amino acid.
Each amino acid has a specific aminoacyl-tRNA synthetase that attaches it to its appropriate
tRNA molecules to create an aminoacyl tRNA.
- The hydrolysis of ATP drives this process.
64 codons for amino acids can be read from mRNA, but there are only 45 different tRNA
molecules.
A phenomenon known as wobble enables the 3rd nucleotide of some tRNA anticodons to pair
with more than one kind of base in the codon.
- One tRNA can recognize more than one mRNA codon, all of which code for the same amino
acid carried by that tRNA.
Ribosomes
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Ribosomes facilitate the specific pairing of tRNA anticodons with mRNA codons during protein
synthesis.
They consist of a large and small subunit, each composed of proteins and a form of RNA called
ribosomal RNA (rRNA)
- The protein components are mostly on the outside and the rRNA is in the interior at the
interface between the subunits and at the A and P sites
Subunits are made in the nucleolus in eukaryotes.
- Prokaryotic ribosomes are smaller and differ enough in molecular composition that some
antibiotics can inhibit them without affecting eukaryotic ribosomes.
A large and small subunit join to form a ribosome when they attach to an mRNA molecule.
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Ribosomes have a binding site for mRNA, a P site that holds the tRNA carrying the growing
polypeptide chain, an A site that holds the tRNA carrying the next amino acid, and an E site (exit
site) from which discharged tRNAs leave the ribosome.
Building a Polypeptide
1. mRNA binds to the small ribosomal subunit (at the 5’ end)
2. The smallest ribosomal unit attaches to mRNA at the “start codon” AUG at the P site
3. The large ribosomal subunit attaches
- It attaches with the aid of proteins called initiation factors, forming the translation initiation
complex
- The initiator tRNA fits into the P site
4. Codon recognition: A charged tRNA brings in an amino acid to the A site
elongation 5. Peptide bond formation: The amino acid released from the tRNA in the P site is transferred to
the amino acid (still attached) to the tRNA in the A site.
- Hydrogen bonds are between tRNA and amino acids  are easily broken
- Covalent bond called a peptide bond forms between amino acids
6. Translocation: tRNA in the A site moves to the P site
7. The uncharged tRNA in the P site moves to the E site
8. The ribosome shifts down to another mRNA codon
9. The site is now open for the next tRNA with an amino acid
10. When the “stop codon” reaches the A site, a release factor binds to the stop codon in the A site
termination
to stop translation
11. The tRNA releases the polypeptide. The ribosome releases mRNA and tRNA and the ribosome
separates.
- The completed polypeptide leaves through the exit tunnel of the large subunit.
- The mRNA strand is broken down afterwards and recycles
12. The polypeptide chain moves through the ER (endoplasmic reticulum)
a) It is folded into its secondary shape
b) Sugars are added as markers
13. Proteins are packaged and shipped in vesicles from the Golgi Apparatus for use outside the cell
initiation
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The start codon is an amino acid (it’s methionine), but the stop codon is NOT an amino acid
The longer the polypeptide chain, the longer the mRNA stays in the cytoplasm before it’s broken
down
Completing and Targeting the Functional Protein
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During and following translation, a polypeptide folds spontaneously into its secondary and
tertiary structures.
- Chaperone proteins often facilitate the correct folding.
The protein may need to undergo post-translational modifications:
- Amino acids may be chemically modified
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- One or more amino acids at the beginning of the chain may be enzymatically removed
- Segments of the polypeptide may be excised
- Several polypeptides may associate into a quaternary structure
All ribosomes are identical, whether they are free ribosomes that make proteins for the cytosol
or make ER-bound ribosomes that make membrane and secretory proteins.
- Polypeptide synthesis begins in the cytoplasm, at ribosomes.
If a protein is destined for the endomembrane system or for secretion, its polypeptide chain will
begin with a signal peptide.
- The signal peptide is recognized by a protein-RNA complex called a signal recognition
particle (SRP), which attaches the ribosome to a receptor protein that is part of a
multiprotein translocation complex on the ER membrane.
- The signal sequence is the directions for the transport of proteins to different parts of the
cell. A sugar molecule is often added to the protein, forming a glycoprotein.
As the growing polypeptide threads into the ER, the signal peptide is usually removed.
Other signal peptides direct some proteins made in the cytosol to specific sites such as
mitochondria, chloroplasts, or the interior of the nucleus
Mutations
Chromosome Mutations
Aneuploid = too many or too few chromosomes
1.
2.
3.
4.
Nondisjunction – when homologous chromosomes don’t separate during meiosis
Deletion – whole or part of the chromosome is deleted
Insertion – whole or part of the chromosome is inserted
Inversion – within a chromosome; when fragments rejoin to the original chromosome in the
reverse orientation
5. Translocation – part of a chromosome gets stuck on another nonhomologous chromosome
Gene Mutations
1) DNA replication
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DNA polymerase proofreads for errors
Excision repair corrects mutations
This reduces the mistake ratio from 1 in 10,000 to 1 in 1 billion
2) Transcriptional errors
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DNA  mRNA
Heterochromatin – not transcribed because it is tightly condensed with the protein
Euchromatin – transcribed
Introns vs. Exons
-Mutations in introns are cut out, so they are not important. But mutations in exons are
expressed.
Failure to add cap/tail  broken down by enzymes
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3) Translational errors
- Point mutations – change in a single nucleotide
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-Example: Sickle cell anemia
Substitutions – may or may not result in a mutation; can cause the wobble effect (=a
change in the 3rd nucleotide is less likely to cause a mutation)
Deletion
Frameshift mutation: causes a shift in the 3 nucleotides of codons that are read
Insertion
Nonsense mutation = when a substitution results in premature stop codon, which stops the
whole message.
Early vs. late in mRNA – the earlier the mutation, the more problematic and dangerous
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