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II. James Watson and Francis Crick discovered the structure of DNA in 1953 by building models.
A. James Watson got the chance to see an X-ray diffraction image of DNA produced by Rosalind Franklin (who worked in
the lab of Maurice Wilkins). That image provided the following clues about the structure of the DNA molecule:
1. It was in the general shape of a helix (corkscrew).
2. The molecule had a uniform width of 2 nm (which suggested that the helix was made up of two strands).
3. The bases were stacked 0.34 nm apart.
B. Using this information, Watson and Crick began constructing molecular models to fit the data.
1. They made a double helix that had the sugar-phosphate chains toward the outside of the molecule and the
nitrogenous bases toward the center.
2.
They oriented the sugar-phosphate backbones in opposite directions (the 5' end of one chain was next to the 3'
end of the other). This is called an antiparallel arrangement.
3. Each base had chemical side groups that allowed it to form hydrogen bonds with its partner on the other strand.
There are two H-bonds between A and T, and three between G and C. (A purine had to pair with a pyrimidine
in order to maintain the uniform width of the molecule throughout.)
C. DNA structure:
1. DNA is a polymer of nucleotides.
2. Each nucleotide is composed of three components:
a. A phosphate group (phosphoric acid)
b. A 5 carbon sugar (deoxyribose in DNA, ribose in RNA)
c. A nitrogenous base (purines = adenine and guanine, pyrimidines = thymine and cytosine). The
pyrimidine uracil replaces thymine in RNA.
3. The carbon atoms of the sugar are numbered starting from the one at the right of the oxygen and moving
clockwise around the molecule. The prime symbol (‘) is used to distinguish the carbons on the sugar from the
carbons on the base.
4. The phosphate group is on the 5' carbon, the base is on the 1' carbon, and a free hydroxyl (OH) group is on the
3' carbon of the sugar.
5. The 5' phosphate and the 3' hydroxyl on different nucleotides can have a covalent bond formed between them
(called a phosphodiester bond). The chain will always have a free 5' phosphate group at one end and a free 3'
hydroxyl group at the opposite end.
I. Gene expression is the process by which DNA directs the synthesis of proteins (or, in some cases, just RNAs).
II. Transcription and translation are the processes that connect genes to proteins.
A. Transcription is the process by which the information stored in the DNA is transcribed into an intermediary molecule of
RNA (i.e. it is the synthesis of RNA under the direction of DNA).
1. All types of RNA are made by transcription; however, the type of RNA that carries the information that is later
used to build a specific polypeptide is called messenger RNA (mRNA).
2. Specific sequences of nucleotides along the DNA mark where transcription of a gene begins and ends.
a. The promoter is the DNA sequence to which the enzyme that catalyzes transcription (RNA
polymerase) attaches and initiates transcription.
1) Bacteria have a single type of RNA polymerase, whereas eukaryotes have at least three
types of RNA polymerases. The one used to synthesize mRNA is called RNA
polymerase II.
b. The terminator is the DNA sequence (in prokaryotes) that signals the end of transcription.
c. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. The
direction of transcription is said to be “downstream”. Therefore, the promoter is upstream from the
terminator.
3. The process of transcription can be divided into three main stages.
a. Initiation is the first event in transcription.
1) The promoter of a gene determines which of the two strands of the DNA helix is used as
the template.
2) In prokaryotes, the RNA polymerase itself specifically recognizes and binds to the
promoter. In eukaryotes, a collection of proteins called transcription factors assists in
the binding of RNA polymerase and the initiation of transcription.
a) Only after certain transcription factors are attached to the promoter does RNA
polymerase II bind to it.
b)A eukaryotic promoter commonly includes a TATA box (a nucleotide sequence
containing TATA, about 25 nucleotides upstream from the transcription start point).
3) Once the RNA polymerase is firmly attached to the promoter DNA, the two DNA strands
unwind there, and the enzyme starts transcribing the template strand.
b. Elongation is the next event in transcription.
1) As the RNA polymerase moves along the DNA, it continues to untwist the double helix,
exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides. (Unlike
DNA nucleotides, RNA nucleotides contain the sugar ribose instead of deoxyribose and
have the pyrimidine base uracil instead of thymine)
2) RNA polymerase adds nucleotides to the 3’ end of the growing RNA chain. Therefore,
transcription (like DNA replication) occurs in a 5’ to 3’ direction.
3) In the wake to transcription, the DNA double helix re-forms and the new RNA molecule
peels away from its DNA template.
4) A single gene can be transcribed simultaneously by several molecules of RNA
polymerase, thus increasing the amount of RNA transcribed from it. This assists the cell
in making large amounts of the encoded protein.
c. Termination is the last step in transcription.
1) In prokaryotes, transcription proceeds until the RNA polymerase transcribes a terminator
sequence in the DNA. The transcribed terminator (existing as an RNA sequence)
functions as the actual termination signal, causing the polymerase to detach from the
DNA and release the transcript (which is available for immediate use as mRNA).
2) In eukaryotes, RNA polymerase II transcribes a sequence of the DNA called the
polyadenylation signal sequence, which codes for a polyadenylation signal (AAUAAA)
in the pre-mRNA. At a point about 10 to 35 nucleotides downstream from the AAUAAA
signal, proteins associated with the growing RNA transcript cut it free from the
polymerase, releasing the pre-mRNA. However, the polymerase proceeds for hundreds
of nucleotides past the site where the pre-mRNA was released.
4. Eukaryotic cells modify the pre-mRNA after transcription.
a. The RNA that is produced initially from the transcription of a eukaryotic gene is called pre-mRNA
(and is also referred to as the primary transcript).
b. Both ends of a pre-mRNA molecule are modified before it leaves the nucleus.
1) The 5’ end is immediately capped off with a modified form of a guanine nucleotide (the
5’ cap). The 3’ end has a poly-A-tail (consisting of 50 to 250 adenine nucleotides)
added to it by an enzyme. The 5’ cap and the poly-A-tail seem to facilitate the export of
the mature mRNA from the nucleus. They also help to protect the mRNA from
degradation by hydrolytic enzymes and help ribosomes attach to the 5’ end of the mRNA
once it reaches the cytoplasm.
c. The most remarkable type of RNA processing in the eukaryotic nucleus is the removal of a large
portion of the RNA that is initially synthesized. This type of modification is called RNA splicing.
1) Most eukaryotic genes have long noncoding stretches of nucleotides interspersed between
coding segments of the gene. Therefore, the sequence of DNA nucleotides that codes for
a eukaryotic polypeptide is not continuous.
a) The noncoding segments are called intervening sequences or introns.
b) The coding regions that are eventually expressed as a polypeptide are called
exons.
2) The introns are cut out of the pre-mRNA molecule and the exons are spliced together to
form the mature mRNA with a continuous coding sequence.
a) The signals for RNA splicing are sets of a few nucleotides located at either
end of each intron. Particles called small nuclear ribonucleoproteins
(snRNPs) ("snurps") play a key role in RNA splicing. snRNPs are made of
proteins and another type of RNA called small nuclear RNA (snRNA).
b) snRNPs carry out their roles as part of a larger, more complex assembly called
a spliceosome. The spliceosome interacts with the ends of an RNA intron and
cuts at specific points to release the intron and then directly joins the two
exons that are now adjacent.
c) RNA splicing in some ciliates such as Tetrahymena has been demonstrated to
occur without proteins or extra RNA molecules. The intron RNA itself
actually catalyzes the process. Therefore, some RNA molecules can function
as enzymes and are called ribozymes.
3) Introns may play regulatory roles in the cell. Also, a number of genes are known to give
rise to two or more different polypeptides, depending on which segments are treated as
exons during RNA processing (this is called alternative RNA splicing).
5. mRNA carries information in the form of a triplet code. Groups of three nucleotides on an mRNA molecule are
called codons and each codon codes for a specific amino acid (i.e. each codon specifies which one of the 20
amino acids will be incorporated at the corresponding position along a polypeptide).
a. The four different kinds of RNA nucleotides can be grouped into groups of three in 64 different
ways (43). Since there are only 20 different kinds of amino acids commonly found in proteins, there is
considerable redundancy in the genetic code. However, the redundancy is not altogether random. In
many cases, codons that are synonyms for a particular amino acid differ only in the third base of the
triplet.
b. Codons must be read in the correct reading frame (i.e. reading the correct groups of three). The
phrase “the fat cat ate the rat” is correctly understood by reading groups of three letters starting with
the first letter. However, if groups of three letters were read starting with the second letter, a nonsense
phrase would be formed (hef atc ata tet her at).
A. Translation is the process by which the information stored in the mRNA is used to produce a specific polypeptide. It
requires the use of transfer RNA (tRNA) molecules which act as interpreters between the nucleic acid language and the
protein language.
1. The function of tRNA is to transfer amino acids from the pool of amino acids in the cytoplasm to a ribosome,
which adds them to the growing end of a polypeptide chain.
2. Each tRNA molecule is used repeatedly, picking up its designated amino acid in the cytosol, dropping it off at
the ribosome, and then leaving the ribosome to pick up another of the same kind of amino acid.
3. tRNA molecules are made of a single strand of RNA that folds back on itself forming hydrogen bonds between
complementary bases in certain regions. Each tRNA molecule has a three-dimensional structure that is roughly
L-shaped with a loop protruding at the upper end of the L. This loop includes a group of three nucleotides
called the anticodon which pairs with a complementary codon on a mRNA molecule during translation.
4. The 3’ end of the tRNA molecule protrudes from the other end of the L. This protruding 3’ end is the site of
attachment for the amino acid that is carried by the tRNA.
a. The reactions by which the correct amino acids are placed on the correct tRNA molecules are
catalyzed by a group of enzymes called aminoacyl-tRNA synthetases (amino acid activating
enzymes). There are 20 of these enzymes, one for each type of amino acid.
b. Surprisingly, the aminoacyl-tRNA synthetases do not recognize the anticodons of the tRNA molecules,
but rather another part of the tRNA molecule. This recognition is said to be determined by a "second
genetic code".
c. The synthetase catalyzes the covalent attachment of the amino acid to its tRNA in a process that is
driven by the hydrolysis of ATP.
5. Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during the formation of a
polypeptide.
a. A ribosome is made up of two subunits (called the large and small subunits). The subunits are
composed of proteins and ribosomal RNA (rRNA) molecules and are assembled in a nucleolus (in
eukaryotic cells).
1) In both bacteria and eukaryotes, large and small subunits join to form a function
ribosome only when they attach to an mRNA molecule.
2) Ribosomes of eukaryotes are slightly larger and differ somewhat from bacterial
ribosomes in the molecular composition. Therefore, certain antibiotic drugs can
inactivate bacterial ribosomes without inhibiting the ability of eukaryotic ribosomes to
make proteins.
b. In addition to a binding site for mRNA, each ribosome has three binding sites for tRNA.
1) The P site (peptidyl-tRNA site) holds the tRNA which is carrying the growing
polypeptide chain.
2) The A site (aminoacyl-tRNA site) holds the tRNA which is carrying the next amino acid
to be added to the chain.
3) The E site (exit site) releases tRNAs after they have discharged their amino acids.
c. The ribosome holds the tRNA and mRNA close together and positions the new amino acid for addition
to the carboxyl end of the growing polypeptide. It then catalyzes the formation of the peptide bond (a
covalent bond) between the amino acids.
d. Ribosome structure strongly supports the hypothesis that rRNA, not protein, carries out the ribosome’s
functions. Thus a ribosome can be regarded as a huge ribozyme. The proteins are largely on the
exterior, apparently playing mainly a structural role.
6. Like transcription, translation also occurs in three main stages.
a. Initiation brings together mRNA, a tRNA bearing the first amino acid of the polypeptide
(methionine), and the two subunits of a ribosome.
1) The small ribosomal subunit attaches to the leader segment at the 5’ (upstream) end of
the mRNA.
2) Downstream on the mRNA is the initiation (start) codon, which has the sequence AUG
and signals the start of translation.
3) The initiator tRNA (which carries the amino acid methionine) attaches to the initiation
codon.
4) The union of the mRNA, initiator tRNA, and a small ribosomal subunit is followed by
the attachment of a large ribosomal subunit, completing a translation initiation complex.
a) This process requires several proteins called initiation factors along with GTP
as an energy source. It also determines exactly where translation will begin
and establishes the reading frame.
5) At the completion of the initiation process, the initiator tRNA sits in the P site of the
ribosome, and the vacant A site is ready to accept the next tRNA molecule.
b. Elongation is the stage of translation in which amino acids are added one by one to the preceding
amino acid. Each addition requires the participation of several proteins called elongation factors and
occurs in a three-step cycle.
1) In the first step of elongation (codon recognition), the mRNA codon in the A site of the
ribosome forms hydrogen bonds with the anticodon of an incoming tRNA molecule
carrying the appropriate amino acid. This step requires the hydrolysis of two molecules
of GTP for energy.
2) In the second step (peptide bond formation), a rRNA molecule of the large ribosomal
subunit (functioning as a ribozyme) catalyzes the formation of a peptide bond that joins
the polypeptide extending from the P site to the newly arrived amino acid in the A site.
a) In this step, the polypeptide separates from the tRNA to which it was attached,
and the amino acid at its carboxyl end bonds to the amino acid carried by the
tRNA in the A site.
3) In the third step of elongation (translocation), the ribosome moves (translocates) the
tRNA is the A site, along with its attached polypeptide, to the P site. The tRNA that was
in the P site is moved to the E site and from there it leaves the ribosome.
a) The codon and anticodon remain hydrogen-bonded, which allows the mRNA
and tRNA to move as a unit. This movement bring into the A site the next
codon to be translated.
b) This translocation step also requires energy in the form of GTP. The mRNA
is moved through the ribosome in the 5' 3' direction only.
c. Termination is the final stage of translation. Elongation continues until a termination (stop) codon
enters the A site. These special codons (UAA, UAG, UGA) do not code for amino acids, but rather
signal the end of translation.
1) A protein called a release factor binds directly to the stop codon in the A site. The
release factor causes the addition of a water molecule instead of an amino acid to the end
of the polypeptide chain. This reaction frees the completed polypeptide from the tRNA
in the P site, thereby freeing the polypeptide from the ribosome.
7. During and after translation, a polypeptide spontaneously coils and folds to assume its characteristic three-
dimensional shape. Disulfide bonds may form between certain cysteine residues. Often, one or more amino
acids at the amino terminal of the polypeptide are enzymatically removed or the polypeptide may be cleaved in
several pieces. Certain amino acids may be chemically modified, for example by the addition of sugars or
phosphate groups. Finally, several identical or different polypeptides may come together to form a quaternary
structure.
8. Typically, a single mRNA is used to make many copies of a polypeptide simultaneously, because a number of
ribosomes work on translating the message at the same time. Such strings of ribosomes are called
polyribosomes. They help a cell to make many copies of a polypeptide very quickly.
9. Free ribosomes are suspended in the cytosol and mostly synthesize proteins that dissolve in the cytosol and
function there. In contrast, bound ribosomes are attached to the endoplasmic reticulum and make proteins
intended for incorporation into the endomembrane system (i.e. nuclear envelope, ER, Golgi apparatus,
lysosomes, vacuoles, and plasma membrane) as well as proteins destined for secretion from the cell.
a. The proteins destined for the endomembrane system or for secretion are marked by a signal peptide,
which targets the protein to the ER. The signal peptide (a sequence of about 20 amino acids at or near
the amino end of the polypeptide) is recognized as it emerges from the ribosome by a protein-RNA
complex called a signal-recognition particle (SRP).
b. The SRP functions as an adapter that brings the ribosome to a receptor protein built into the ER
membrane. Polypeptide synthesis continues there, and the growing polypeptide is threaded across the
ER membrane and into the cisternal space via a protein pore. The signal peptide is usually removed by
an enzyme.
III. A mutation is a change in the genetic material of a cell. It can involve large portions of a chromosome (chromosomal
mutation) or as little as one base pair (point mutation).
A. If a point mutation occurs in a gamete or in a germ-line cell (i.e. a cell that gives rise to gametes), it may be transmitted
to offspring and to a succession of future generations. However, mutations affecting somatic cells are not heritable.
B. Types of point mutations:
1. Base pair substitution is the replacement of one nucleotide and its partner from the complementary DNA
strand with another pair of nucleotides. Depending on how a base-pair substitution is translated via the genetic
code, it may result in no change in the protein encoded by the mutated gene (a so-called silent mutation), in an
insignificant change in that protein, or in a noticeable alteration (which may be critical to the life of the
organism).
a. Missense mutations occur when the mutated codons still code for amino acids, just not the correct
ones.
b.Nonsense mutations occur when a amino acid-specifying codon is changed to a stop codon. This
results in a polypeptide that is shorter than the one coded for by the normal gene, and is therefore
nonfunctional most of the time.
2. An insertion or deletion of one or more nucleotide pairs in a gene results in a frameshift mutation. All of the
nucleotides downstream of the mutation will be incorrectly grouped into codons, and the results will be
extensive missense ending sooner or later in nonsense (which causes premature termination of translation).
Unless a frameshift occurs very near the end of the gene, it is almost certain to result in a nonfunctional protein.
C. Mutagens are physical or chemical agents that interact with DNA to cause mutations.
1. Ionizing radiation is high energy radiation such as X-rays and gamma rays. This type of radiation causes
double-stranded breaks in the DNA that can result in chromosomal rearrangements and deletions.
2. Ultraviolet radiation causes the formation of thymine dimers. The repair process for thymine dimers is error
prone, and may result in base-pair substitutions, insertions, or deletions.
3. Base analogues are chemicals that resemble normal bases, but pair incorrectly during DNA replication. They
can cause base-pair substitutions.
4. Intercalating chemicals stack between the bases of the double helix and distort it. They can lead to small
insertions or deletions during DNA synthesis (and so cause frameshift mutations).
5. Other mutagens cause chemical changes in bases and change their pairing properties.
D. Recombination of genes on chromosomes can also result in mutations.
1. Transposons (so-called jumping genes) are segments of DNA that occasionally move out of one DNA site and
into another. They can act as mutagens by disrupting the function of the gene into which they insert (called
insertional inactivation). They can also cause deletions by carrying adjacent nucleotides with them.
2. Unequal crossing over is caused by improper pairing of homologous chromosomes during prophase I. This
results in deletions and insertions.
3. Gene conversion can occur when homologous chromosomes are non-identical (i.e. carry different alleles at a
particular locus). During synapsis, DNA repair enzymes can detect the mispairing between the non-identical
regions of the homologues. One of the mismatched strands is excised and the gap filled in a way that is
complementary to the other strand. This results in two chromosomes now having the same sequence; one of the
two mismatched sequences has been lost.
IV. Archaea share many aspects of the mechanisms of gene expression with eukaryotes, as well as a few with bacteria.
A. The single RNA polymerase of archaea resembles the three eukaryotic RNA polymerases and archaea (like eukaryotes)
use a complex set of transcription factors.
B. Archaeal ribosomes are the same size as bacterial ribosomes, but their sensitivity to antibiotics most closely matches that
of eukaryotic ribosomes.
C. Introns are present in some of the genes of archaea, but are very rare in bacteria. Introns are present in eukaryotic genes.