UNIT 1 – STRUCTURE
DNA vs RNA
-DNA has thymine (which has a methyl group), while RNA has Uracil (no methyl group)
-DNA has a ribose (H), RNA has a deoxyribose (OH).
Function of protein in DNA: to neutralize the negative charge caused by a negative phosphate
Frederick Griffith Experiment
He worked with two forms of Streptococcus pneumoniae. The smooth strain—S—has a
polysaccharide capsule surrounding each cell, and is virulent. The rough strain—R—does not
have a polysaccharide capsule , and is nonvirulent. Therefore the capsule is responsible for the
virulence of the S strain.
-killed S bacteria and injected into mice, they lived
-heat-killed S and live R forms mixed together and injected into mice, the mice died.
Therefore the live R forms must have been transformed into S forms by acquiring the ability to
make a polysaccharide capsule.
Later it was found out by Oswald Avery that DNA was the transforming agent.
He used the same bacteria as Griffith, and mixed heat-killed S forms with R form. Then, when
he killed off protein or RNA in the cells, the R form was still being transformed into S form. This
meant that DNA was the transforming agent.
Hershey and Chase
They worked with bacteriophages (viruses that infect cells) because a virus has DNA/RNA
inside its protein coat. They tagged DNA and the protein separately with radioactive labels.
- DNA entered the cells
- Protein coat remained on outside
Therefore, DNA was the transforming agent.
Watson and Crick
DNA contains 4 nucleotides (ATGC)
DNA is made of 5 carbon sugar (deoxyribose) + phosphate group +1 of the four nitrogenous
bases – ATGC
-A and G are purines (2 rings)
-T and C are pyrimidines (1 ring) -purines bind with pyrimidines (A-T, G-C) via hydrogen bonding
-Erwin Chargaff (A=T, G=C)
- DNA has a sugar-phosphate backbone because the nucleotides in one strand
(polynucleotide) are joined by phosphates.
-5’ end = phosphate group, 3’end = hydroxyl group; strands are antiparallel
- Rosalind Franklin used x-ray diffraction to determine that the DNA has a helical
structure. She could also tell the the DNA is 2nm in diameter and patterns of atom repeats
occurred every 0.34 nm (1 base pair) and 3.4 (1 full helix turn) nm in length.However, she did
not have crystals to work with which would have made her results more accurate.
Meselson and Stahl
- DNA is semiconservative
Procedure: Used “heavy” nitrogen isotopes to label the parent strands of the DNA. They
transferred the N-15 labeled DNA into a “light” nitrogen isotope medium (N-13) so that the newly
synthesized DNA strands would incorporate it.
UNIT 2 1 DNA Replication
DNA Polymerase assembles the complementary strands of DNA from individual nucleotides.
More than one kind of DNA polymerase is required for DNA replication in both eukaryotes and
prokaryotes. Nucleoside triphosphates (nitrogenous base+deoxyribose
sugar+3phosphates=dATP, dGTP, dCTP,dTTP) are substrates for DNA polymerase. DNA
polymerase can only add nucleotides to the 3’end, therefore the new strand is synthesized in
the 5’ 3’ but is read from 3’5’.
DNA helicase unwinds the DNA by using energy from ATP hydrolysis.
Single-stranded binding proteins stabilize the DNA for replication so that the unwound DNA
does not twist back together.
Topoisomerase prevents the DNA from overtwisting
Primase lays on the primer so that DNA polymerase can begin synthesis. Later, the primer is
removed and replaced by DNA.
Leading Strand is synthesized continuously in the direction of unwinding.
Lagging Strand is synthesized discontinuously in “okazaki fragments”
DNA Polymerase I removes the RNA primer
DNA Ligase joins the space between okazaki fragments and closes the nicks. When the initial primer is removed, a gap is left which is not replaced by DNA. This means that
with each replication of the DNA, the complementary strand becomes shorter and shorter and
eventually this can become lethal for the cell. In most cases, there is a buffer of noncoding DNA
at the ends of the strand so as to protect it – called Telomeres (TTAGGG in humans). The
enzyme Telomerase helps rebuild the telomeres. Telomerase is not active in all cells. It is active
in sperm and egg cells, but not in mature cells. This means the cell eventually dies and is
replaced by another one. Therefore, the shortening of telomeres = aging, and constantly active
telomeres = cancer.
Replication Origins are regions where replication begins. The replication origins are only
activated once during S phase as to prevent the same parts of a gene from being replicated
Proofreading mechanism is a mechanism that proofreads the DNA polymerase’s mistakes.
When a mispaired nucleotide is added, the DNA polymerase cannot move forward so it reverses
and replaces the nucleotide with the correct one.
Any base-pair mismatches that remain after the DNA polymerase has proofread itself are
repaired by the DNA repair mechanism. The repair enzymes move along newly replicated DNA
molecules, “scanning” the DNA for distortions in the newly synthesized nucleotide chain. If the
enzymes encounter a distortion, they remove a portion of the new chain, including the
mismatched nucleotides. The gap left by the removal (step 2) is then filled by a DNA
polymerase, using the template strand as a guide (step 3). The repair is completed by a DNA
ligase, which seals the nucleotide chain into a continuous DNA molecule (step 4) UNIT 2-2 Transcription
Transcription is the mechanism by which the information encoded in DNAis made into a
Transcription is the first step in a process whereby particular genes are expressed in any given cell at a
given time. Some of those genes are protein-coding genes that encode mRNAs to be translated; others are
non–protein-coding genes that encode RNAs that are never translated, such as ribosomal RNAs (rRNAs),
transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs).
→ RNApolymerase creates the complementary RNAsequence following complementary base
→ The RNAtranscribed from a gene encoding a polypeptide is called messenger RNA(mRNA).
Process Prokaryote Eukaryote
Transcription and Translation Occur at the same time. No Separate processes
pre-mRNA (transcription in nucleos,
translation in cytoplasm)
Initiator codon AUG AUG
Promoter In prokaryotes, the promoters
The promoters of protein-
are immediately upstream of coding genes are immediately
where transcription initiates upstream of the transcription
start point and are typically
more complex than in
factors bind to the TATA box
and recruit RNApolymerase # RNApolymerases Since all the other types of different polymerases for
genes in prokaryotes (for transcribing different types of
example, tRNAand rRNA genes. RNApolymerase II
genes) have similar promoters, transcribes protein-coding
the same RNApolymerase genes. RNApolymerases I and
complex can transcribe them III transcribe genes for non-
all. protein-coding RNAs.
Termination are two types of specific DNA no equivalent “transcription
sequences called terminators terminator” sequences. The 3′end
that signal the end of of the gene is a sequence that is
transcription of the gene. Both to be transcribed into the pre-
types of terminator sequences mRNA. Proteins bind to
act after they are transcribed. Inthis polyadenylation signal and
the first case, the terminator cleave the pre-mRNA at that
sequence on the mRNAbase- point. This signals the RNA
pairs with itself to form a polymerase to stop transcription.
“hairpin.” In the second case, a
protein binds to the terminator
sequence on the mRNA. Both of
these mechanisms trigger the
termination of transcription and
the release of the RNAand RNA
polymerase from the template.
→ Genetic code – nucleotide information that specifies that aa sequence of a polypeptide
→ Codon – three bases that “code” for a word. By convention, scientists write the codons in the
5′→ 3′ direction as they appear in mRNAs.
o Initiator codon – AUG (methionine) is the first codon translated in any mRNA
o One of these codons,AUG, specifies the amino acid methionine. It is the first codon
translated in any mRNAin both prokaryotes and eukaryotes.
o Stop codon (nonsense or termination codons) – (UAA, UAG, UGA) doesn’t specify an
aa and that act as indicators of the end of the polypeptide sequence
Only two amino acids, methionine and tryptophan, are specified by a single codon. All the rest are
represented by at least two, some by as many as six. In other words, there are many synonyms in the
nucleic acid code, a feature known as degeneracy (or redundancy). For example, UGU and UGC both
specify cysteine, whereas CCU, CCC, CCA, and CCG all specify proline. → There is only one reading frame for each mRNA(have to start at correct initiator codon and read
3 bases at a time)
→ The code is also universal. With a few exceptions, the same codons specify the same amino acids
in all living organisms, and also in viruses.
SET RULES OF TRANSCRIPTION:
• in a given gene, only one of the two DNAnucleotide strands acts as a template for
synthesis of a complementary copy, instead of both, as in replication.
• only a relatively small part of a DNAmolecule—the sequence encoding a single gene—
serves as a template, rather than all of both strands, as in DNAreplication.
• RNApolymerases catalyze the assembly of nucleotides into an RNAstrand, rather than
the DNA polymerases that catalyze replication.
• the RNAmolecules resulting from transcription are single polynucleotide chains, not
double ones, as in DNAreplication.
STEPS IN TRANSCRIPTION
1. helicase unwinds the DNA
2. DNApolymerize can start synthesis WITHOUT a primer, by binding to a promoter
region and synthesizing in the 5’-3’direction while reading the strand 3’-5’.
3. The part of the gene that is to be transcribed into RNAis called the transcription unit.
4. DNApolymerase reaches a terminating sequence and the completed RNAtranscript and
RNApolymerase are released from the DNA
Pre-mRNAhas exons and introns, while mRNAonly has exons.
Introns = noncoding
Exons = coding
Aeukaryotic protein-coding gene is typically transcribed into a precursor-mRNA (pre-mRNA) that
must be processed in the nucleus to produce translatable mRNA. The mature mRNAexits the nucleus and
is translated in the cytoplasm.
MODIFICATIONS At the 5′ end of the pre-mRNAis the 5′ cap, consisting of a guanine-containing nucleotide that is
reversed so that its 3′-OH group faces the beginning rather than the end of the molecule.Acapping
enzyme adds the 5′cap to the pre-mRNA. The cap, which is connected to the rest of the chain by three
phosphate groups, remains when pre-mRNAis processed to mRNA. The cap functions as the initial
attachment site for mRNAs to ribosomes to allow translation.
The termination of transcription of a eukaryotic protein-coding gene is different from that of a prokaryotic
gene in that there is no terminator sequence at the end of the gene in the DNA. Instead, at the 3′end of the
gene is a sequence that is to be transcribed into the pre-mRNA. Proteins bind to this polyadenylation
signal and cleave the pre-mRNA at that point. This signals the RNApolymerase to stop transcription.
Then the enzyme poly(A) polymerase adds a chain of 50 to 250 adenine nucleotides, one nucleotide at a
time, to the newly created 3′end of the pre-mRNA. The string of adenine nucleotides, called the poly(A)
tail, enables the mRNA produced from the pre-mRNAto be translated efficiently and protects it from
attack by RNA-digesting enzymes in the cytoplasm.
Aprocess called mRNAsplicing, which occurs in the nucleus, removes introns from pre-mRNAs and
joins exons together. How does this occur? mRNAsplicing occurs in a spliceosome, a complex formed
between the pre-mRNA and a handful of small ribonucleoprotein particles (snRNPs)
Alternative Splicing. The removal of introns from a given gene is not absolute. That is, in certain tissues,
or under certain environmental conditions, exons may be joined in different combinations to produce
different mRNAs from a single DNAgene sequence. The mechanism, called alternative splicing, greatly
increases the number and variety of proteins encoded in the cell nucleus without increasing the size of the
genome. This process produces different mRNAs fro the same pre-mRNAby removing different
combinations of exons along with the introns.
Exon Shuffling. different exons either within a gene or between two nonallelic genes are occasionally
mixed. Evolution of new proteins by this mechanism would produce changes much more quickly than by
changes in individual amino acids at random points.
UNIT 2-3 TRANSLATION
Translation is the use of the information encoded in the RNAto assemble amino acids into a polypeptide.
Translation Occurs all over cell Occurs mostly in cytoplasm
mRNAavailability Available right away because it’s Must first exit the nucleus
not confined to a nucleus
Translation initiation the rRNAof the ribosomal rRNAscans the mRNAfrom 5’–
subunit finds the region with the 3’searching for the start codon
start codon directly by base
pairing with a specific ribosome
binding site on the mRNAjust upstream of the start codon. The
large ribosomal subunit then
binds to the small one to
complete the ribosome.
Elongation 15-20 times/sec 1-3 times/sec
→ In translation, an mRNAassociates with a ribosome, a particle on which amino acids are linked
into polypeptide chains. As the ribosome moves along the mRNA, the amino acids specified by
the mRNAare brought by tRNAs and joined one by one to form the polypeptide encoded by the
→ All tRNAs can base-pair with themselves to wind into four double-helical segments, forming a
cloverleaf pattern in two dimensions.At the tip of one of the double-helical segments is
the anticodon, the three-nucleotide segment that pairs with a codon in mRNAs.At the other end
of the cloverleaf is a double-helical segment that links to the amino acid corresponding to the
→ wobble hypothesis proposed that the complete set of 61 sense codons can be read by fewer than
61 distinct tRNAs because of the particular pairing properties of the bases in the anticodons. That
is, the pairing of the anticodon with the first two nucleotides of the codon is always precise, but
the anticodon has more flexibility in pairing with the third nucleotide of the codon.
→ The process of adding an amino acid to a tRNAis called aminoacylation(literally, the addition of
an amino acid) or charging (because the process adds free energy as the amino acid–tRNA
combinations are formed). The final tRNAwith an amino acid is called an aminoacyl–tRNA.
Enzymes called aminoacyl–tRNAsynthetases catalyze aminoacylation
→ Ribosomes Are rRNA–Protein Complexes that Work as Automated ProteinAssembly Machines.
They translate the mRNAinto a sequence of amino acids.
→ Chloroplasts and mitochondria each have their own ribosomes in addition to those in the
Structure of ribosomes:
→ Afinished ribosome is made up of two parts of dissimilar size, called the large and small
ribosomal subunits → The Asite (aminoacyl site) is where the incoming aminoacyl–tRNA(carrying the next amino
acid to be added to the polypeptide chain) binds to the mRNA. The Psite (peptidyl site) is where
the tRNAcarrying the growing polypeptide chain is bound. The E site (exit site) is where an
exiting tRNAbinds as it leaves the ribosome.
STEPS OF TRANSLATION
1. Initiation - assembly of all the translation components on the start codon of the mRNA. In
translation initiation, a large and small ribosomal subunit associates with an mRNA molecule and
the first aminoacyl–tRNAof the new protein chain becomes bound to theAUG start codon. This
is now called the initiator tRNA with an anticodon to the methionine-specifyingAUG start
codon. Each step in translation initiation is aided by proteins called initiation factors.
→ The complex binds to the mRNAat the 5′cap and then moves along the mRNA—a
process called scanning—until it reaches the firstAUG codon (step 2). This is the start
codon, and it is recognized by the Met–tRNA's anticodon. The large ribosomal subunit
then binds, completing the ribosome (step 3).At the end of initiation, the initiator Met–
tRNAis in the P site.
2. Elongation - Elongation involves reading the string of codons in the mRNAone at a time while
assembling the specified amino acids into a polypeptide.
→ The P site, with one exception, can only bind to a peptidyl–tRNA—a tRNAlinked to a
growing polypeptide chain containing two or more amino acids. The exception is the
initiator tRNA, which is recognized by the P site as a peptidyl–tRNAeven though it
carries only a single amino acid, methionine. TheAsite can bind only to an aminoacyl–
tRNA. The tRNApreviously in the P site binds to the E