LS1a Fall 2014
Section Week #6
I. DNA replication
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DNA contains the information necessary to encode cellular proteins.
Genetic information flows from DNA to RNA to protein; this concept is commonly referred to as
the Central Dogma of molecular biology.
DNA replication is the process of duplicating the genetic information contained in DNA prior to cell
division.
When double-stranded DNA is replicated, the two antiparallel strands are separated, and a new
complementary strand is polymerized opposite each of the original strands, resulting in two
identical copies of DNA.
Section Activity #1: Shown below is the mechanism for extension during DNA replication.
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γ
a. Which atom in the growing strand is the nucleophile? To which numbered carbon is it
attached?
The oxygen that is bound to the 3’ carbon is acting as the nucleophile.
b. Label the phosphate groups of the nucleoside triphosphate with their Greek letter
designations.
(Continued on next page…)
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Section Activity #1 (continued from previous page):
c.
In which direction (5’3’ or 3’5’) does strand elongation occur? Why does elongation always
occur in this direction?
Elongation occurs in the 5’ to 3’ direction. This is because the active site of DNA
polymerase catalyzes the nucleophilic attack of a 3’ end oxygen of the growing
strand on the alpha-phosphorus of an incoming deoxynucleoside triphosphate. The
5’ end alpha-phosphorus of the deoxynucleoside triphosphate is the electrophile.
d. Use Le Chatelier’s principle to explain how pyrophosphate hydrolysis drives DNA
polymerization.
The polymerization reaction of DNA releases a molecule of pyrophosphate. This
product is cleaved by an enzyme (“pyrophosphatase”) into two inorganic
phosphate groups. Since this effectively removes a product of the DNA synthesis
reaction, the DNA synthesis reaction must shift to the right in order for the ratio of
products to reactants to remain equal to Keq.
e. Draw a circle around the base that is being added. Draw a box around the base that came
before it.
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The replication of DNA requires four components:
o a single-stranded DNA template (the DNA that is being replicated)
o a primer (a piece of DNA or RNA that provide the 3’-OH to which deoxyribonucleotides are
initially added)
o dNTPs (a mixture of each of the four deoxynucleoside triphosphates)
o DNA polymerase (the enzyme that catalyzes the polymerization reaction)
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DNA replication takes place at localized sites on the DNA molecule called replication forks. The
leading strand is synthesized continuously while the lagging strand is synthesized as shorter
discontinuous fragments that are later linked together by an enzyme called DNA ligase.
Section Activity #2: Shown below is a replication bubble, which is a combination of two replication
forks. The 5’ ends of newly synthesized strands have been labeled. Assume that the lagging
strands shown in the diagram have already been ligated together.
a. Label the 5’ and 3’ ends of both template strands.
b. Using arrows, label the direction in which each replication fork moves.
c.
Label the leading and lagging strands for each replication fork. Use arrows to label the
directions in which each newly synthesized strand is extended.
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II. Polymerase chain reaction (PCR)
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Researchers use DNA polymerases to replicate and amplify DNA in vitro using a technique called
PCR (polymerase chain reaction). This technique enables the exponential amplification of a
specific region of DNA.
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The requirements for PCR are very similar to the components required for cellular DNA replication:
a DNA template, single-stranded primers, dNTPs, and a DNA polymerase that can be functional at
high temperatures.
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There are three steps in a PCR reaction that are repeated for 30-40 cycles:
1. Denaturation (usually at ~94°C): The high temperature denatures the double-stranded DNA
into single strands, providing the single-stranded template for DNA polymerase.
2. Annealing (temperature varies based on the primers, but it usually ranges from 50-70°C):
The primers hybridize with the single-stranded template. Primers that match the DNA exactly
will form stable hydrogen bonds that allow the primer to bind.
3. Extension (usually at 72°C): The DNA polymerase extends the primer, incorporating dNTPs
that are complementary to the template.
Section Activity #3: Shown below is a diagram of a segment of linear double stranded DNA. The
segment is divided into four regions, A, B, C, and D. While the DNA sequences for each region are
not shown, the DNA sequences at the boundaries between each region in the top (solid) strand
are given.
Two 8-nucleotide primers, 5’-AGTTGCGA-3’ and 5’-TCCACGGG-3’, were added to a PCR reaction to
amplify the DNA shown above. What are the products that would result in vast excess after the
many rounds of PCR? Indicate a product as either a dotted or solid line, depending on whether its
sequence matches that of the dotted or solid strand, respectively. Label the 5’ and 3’ ends of the
product strands and include the primer sequence at the 5’ end. Also indicate which regions are
contained in each strand.
Answer:
III. Transcription
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DNA strands can be named depending on whether or not they act as a template for transcription.
The “template” strand is complementary to the RNA that is transcribed, while the “non-template,”
or “coding,” strand has the same sequence as the RNA that is transcribed (except the DNA code
includes T instead of U).
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The enzyme RNA polymerase initiates transcription at specific sequences called promoters.
Promoters are regions of DNA that:
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o
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Act as the binding sites for RNA polymerase
Mark the start of a given gene
-35
Define the direction of RNA transcription
IIIa. Transcription in Prokaryotes (Bacteria)
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Initiation: Promoters in prokaryotes contain recognition sequences around the -10 and -35
positions. These are consensus sequences that are recognized by sigma factor (σ), a subunit of
the bacterial RNA polymerase. The binding of the RNA polymerase (“RNA Pol”) to these sequences
places the polymerase in the appropriate location with the appropriate orientation to initiate
transcription.
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When RNA polymerase initially binds to the promoter, the transcription bubble is not yet
opened, and so the enzyme-DNA complex is referred to as a “closed” complex.
o
RNA polymerase next unwinds a short stretch of DNA (~13 bases long), opening up a
transcription bubble, and generating an “open” enzyme-DNA complex.
o
Transcription initiates when RNA polymerase uses one of the exposed DNA strands in the
transcription bubble as a template to catalyze the formation of the first phosphodiester bond
between two ribonucleotides that are complementary to the +1 and +2 bases of the DNA
template.
During elongation, RNA polymerase continues to synthesize a ribonucleic acid polymer (a
“transcript”) complementary to the DNA template strand. As it does so, the replication bubble
moves with the RNA polymerase as double-stranded DNA is unwound in front of the enzyme and
rewinds behind the moving RNA polymerase. Just like DNA polymerase, RNA polymerase links
ribonucleotides in the 5’ to 3’ direction.
IIIb. Transcription in Eukaryotes
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Eukaryotes use transcription factors (TFs) instead of a sigma subunit to identify the promoter. A
key event is the binding of TATA Binding Protein (TBP) to the TATA box (located ~25-30
nucleotides upstream of the transcription start site).
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Transcription initiation can be influenced by activators and repressors, which can either bind
upstream of downstream of the promoter sequence.
Section Activity #4: Consider the sequence of a bacterial promoter as shown below.
-10
non-template (“coding”)
5’-GCCTATCGACTTACTTGACACGCATCGGACTTAGGTCTATATTGACTTAACTGGATCAAGCT-3’
3’-CGGATAGCTGAATGAACTGTGCGTAGCCTGAATCCAGATATAACTGAATTGACCTAGTTCGA-5’
template
A. Identify the start site of transcription (there is no zero position).
The +1 site is underlined
B. Write the sequence of the first 5 bases of the RNA transcript that is produced and label the 5’ and
3’ ends of the transcript.
5’–ACUGG–3’
C. Label the template and non-template (coding) strands.
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IV. mRNA processing in eukaryotes
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In eukaryotes, pre-mRNA is modified in three ways to form mature mRNA (where “m” =
“messenger”). These modifications do not take place in prokaryotic cells.
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In eukaryotic cells, pre-mRNAs are modified by 5’ capping, 3’ polyadenylation, and splicing in the
nucleus.
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RNA splicing is referred to as a “transesterification” reaction because it involves the transfer of
phosphodiester bonds.
Section Activity #5: Place an X in boxes to indicate whether the features or processes below are
characteristic of prokaryotic cells, eukaryotic cells, or both. [Note to TFs: 1:30]
Section Activity #6: Lab review.
Last week in lab we purified a histidine-tagged protein (GFP) using a porous column filled with
immobilized Ni2+ ions. To do so, we applied the following solutions to the column filled with Ni 2+
ions:
Crude cell lysate
Wash buffer 1
Elution Buffer
Equilibration Buffer
Wash Buffer 2
Arrange the buffers in the appropriate order that they were applied to the Ni2+ column during last
week’s lab.
1. Equilibration buffer
2. Crude cell lysate
3. Wash buffer 1
4. Wash buffer 2
5. Elution buffer
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