Unit 2 - Active Recall Flashcards
Alfred Hersey and Martha Chase - 1952
Demonstrated that DNA not protein is transmitted or
passed on to progeny.
Erwin Chargaff - 1948
Analyzed the nucleotide composition of
DNA - A=T; G=C.
Franklin & Willams - 1953
Watson & Crick devise the secondary structure
Distinguish purines (A&G) from pyrimidines (C&T)?
Purines
- Double carbon-nitrogen ring with four nitrogen atoms
- Bigger
- Adenine and Guanine in both DNA and RNA
Pyrimindines
- Single carbon-nitrogen ring with two nitrogen atoms
- Smaller
- Cytosine in both DNA and RNA, Uracil only in RNA, Thymine only in DNA
Basic structure of the nucleotide?
- BASE A, G, C or T: Attached to sugar 1’ carbon
- PHOSPHATE GROUP: attached to sugar 5’ carbon
- DEOXYRIBOSE SUGAR: 5 carbon sugar OH at 3’ carbon
DNA vs RNA
DNA
- Deoxyribonucleotide
RNA
- Ribonucleotide
- use a RIBOSE sugar
- uses the pyrimidine base uracil instead of thymine
Recall all of the history of the discovery of DNA
- Rosalind Franklin & Maurice Wilkins, using x-ray
diffraction, determined that DNA was a helix of
constant diameter. - Nucleotide structure & Erwin Chargaff’s rule
provided information on base pairing and
positioning of the nucleotides within the helix. - James Watson and Francis Crick used model
building techniques developed by Linus Pauling.
Watson and Crick initially favored a model with
bases on outside. Pauling also favored a base-out
model, but with 3 strands. - Franklin pointed out that phosphates on the
inside would make molecule unstable, hence
phosphates were on the outside. Watson and
Crick using Franklin’s data proposed the structure
for DNA.
What do you know about DNAs double helix structure?
- The backbone of each DNA strand is a repeating deoxyribose sugar-phosphate polymer
- The planar (flat) bases stack on top of each other, perpendicular to the helix axis
- The sequences of bases in the two strands are determined by hydrogen bonding between adenine and thymine or guanine and cytosine
- The B-DNA structure is a right-handed helix with 10 base pairs per rotation of the helix
- Two grooves: major and minor
groove. - Structure does not depend on any particular sequence, structure is more conserved than sequence.
- The strands of DNA are anti-parallel, spiraling around the helix axis in opposite directions
How do you melt or denature DNA? What does this do?
- “Melting” is the separation of two DNA strands.
- Separation is reversible (renature).
- Process allows artificial hybrid molecules to form (strands from a different sources).
- Ways to “melt” or denature DNA: increase temperature; reduce salt concentration; increase pH; solvents
Melting temperature (Tm) of DNA?
- Melting temperature (Tm) by definition is the
temperature at which one half of the DNA duplex will
dissociate to become single stranded and
indicates the duplex stability. - DNA melting or denaturation can be monitored by measuring absorbance.
- As the DNA duplex separates - absorbance increases (hyperchromic shift).
- Tm is an indication of the stability of the hybridized DNA molecule.
- The higher the Tm the more stable the DNA helix.
- The higher the GC content (% G+C), the higher melting
temperature.
Why does salt affect melting temperature?
- The higher the salt concentration of the solution, the higher melting temperature.
- The repulsion between the negatively charged phosphate backbones destabilizes the double helix.
- The negative charges are shielded by salt ions (e.g. Na+) in the solution, this stabilizes the helix and increase
the melting temperature.
What can you do using known melting temperature?
- Classify organism e.g. bacteria, because the GC content in the DNA is species specific.
- Rare gene mutations can be detected because mutated DNA sequences melt at different temperatures than ‘normal’ ranges.
- Process of DNA melting plays an important role in molecular biology techniques, e.g. polymerase chain reaction and southern blotting.
Melting temperature constant?
Tm = 81.5 + 16.6 log[M] + 0.41(%GC) - 675/L
Note:
- M = molar concentration of ions in solvent..example [Na+] = 100 mM = 0.1M
- %GC = percentage of G’s + C’s
- L = length of DNA measured in base pairs (bp)
- Tm depends on ionic strength of buffer, GC content, and length of DNA
Explain the Meselson and Stahl Experiment?
- Grew E. coli on 15N medium for many generations.
- Switched some cells to 14N medium.
- Used equilibrium density gradient centrifugation to
determine isotope composition of DNA.
What is semi-conservative replication?
- Separation of the two DNA strands of the parental molecule.
- Each parental strand serves as a template that determines the order of nucleotides along the
newly synthesized strand. - Each “daughter” DNA molecule consists of one parental strand and one newly synthesized strand
What are the requirements for DNA synthesis?
1) Template of single-stranded DNA (ssDNA)
2) All four deoxyribonucleoside 5’ triphosphates (dNTPs)
3) DNA polymerase and other
enzymes and proteins
4) Free 3’-OH group
What do you recall about DNA polymerase?
- Uses deoxyribonucleoside 5’ triphosphates (dNTPs).
- Catalyze phosphodiester bonds.
- Has an absolute requirement for a preexisting 3’ OH.
- Cannot make a DNA chain de novo. In other words it can only extend a chain
- Always elongates chain in the 5’ to 3’ direction.
- Template strand is always read in the 3’ to 5’
direction.
Describe synthesis at the replication fork.
- Replication begins at a specific nucleotide sequence - origin of replication.
- Synthesis takes place within a replication bubble.
- Both DNA strands are synthesized simultaneously at the replication fork.
- Replication is semidiscontinuous
Compare circular and linear genome modes of replication?
CIRCULAR GENOMES
1. Theta replication (bacteria, e.g. E. Coli)
- Single replicon (for bacteria = entire chromosome).
- Bidirectional replication – two replication forks within a replication bubble.
- Replication is semidiscontinuous at both replication forks.
- Rolling circle replication (viruses)
- No replication bubble.
- Uncoupling of the replication of the two strands of the DNA molecule.
- Replication is continuous
LINEAR GENOMES
3. Linear replication (eukaryotes)
- Multiple replicons, origins of replication, replication bubbles.
- Bidirectional replication – two replication
forks within a replication bubble.
- Replication is semi discontinuous at both
replication forks.
How does DNA replication occur in prokaryotes?
- Initiation
- Initiator proteins bind to the origin of replication (oriC).
- A short section of DNA is unwound and proteins bind to the ssDNA.
- Single-strand-binding proteins keep DNA strands separated.
- Helicase binds to lagging strand template; breaks hydrogen bonds. - Unwinding
- DNA helicase separates the two DNA strands by breaking the hydrogen bonds
- DNA gyrase (a topoisomerase) travels ahead of the
replication fork and alleviates supercoiling caused
by unwinding. - Elongation
- Short stretch of RNA nucleotides (RNA primer) is
synthesized by Primase.
- RNA primer provides a free 3’OH for the DNA polymerase to use.
- The RNA primer is later removed and replaced with DNA nucleotides. - Termination
Overall DNA synthesis
- Connects Okazaki fragments by sealing nicks in the sugar–phosphate backbone.
- Unwinds the double helix by breaking the hydrogen
bonding between the two strands at the replication
fork. - Reduces coiling that builds up ahead of the
replication fork as a result of unwinding. - Binds to origin of replication and causes a short
section of DNA helix to unwind. - Prevents the formation of secondary structures
within single-stranded DNA
DNA primase requires a _____
template and _____ nucleotides to carry out primer
synthesis.
What do you know about E.coli DNA polymerases?
- five DNA polymerase in E. coli (Pol I to Pol V)
(see table 12.3) - All five have 5’ to 3’ polymerase activity.
- Some polymerase have exonuclease activity (to remove
a newly incorporated nucleotide that does not match the
template strand) - Pol III is the principle replication enzyme.
- Pol I removes and replaces RNA primers with DNA.
How are RNA nucleotides replaced?
- DNA Pol I 5’ -> 3’ exonuclease activity removes RNA primers starting at the 5’ ends.
- DNA Pol I 5’ -> 3’ polymerase activity fills in the gap with DNA nucleotides.
- DNA ligase seals the nick in the sugar phosphate
backbone.
What are the three eukaryotic DNA polymerases you need to recall?
DNA polym α: initation of nuclear DNA synthesis and DNA repair (has primase activity)
DNA polym δ: lagging strand synthesis of nuclear DNA, DNA repair, and translesion DNA synthesis
DNA polym ε: leading-strand synthesis
Describe how eukaryotic DNA replication is initiated?
Controlled initiation:
1) An origin must be selected or “licensed” by replication licensing factors.
2) Origin is then activated and replication begins.
3) Once activated/replicated an origin is deactivated.
Note:
- Multiple origins are used during DNA synthesis
- Origins of replication are activated in clusters (20-80
at a time). Each cluster is known as a replication unit.
Explain nucleosome disassembly and reassembly?
- Eukaryotic DNA is packaged into chromatin.
- Need to disassemble, produce more histones and
reassemble nucleosomes.
What is the problem and solution for replication of 3’ ends?
PROBLEM
Telomeres:
- are the ends of linear chromosomes.
- are made up of G-rich short repeated sequence.
- stabilize chromosomes.
Note:
- Each round of replication leaves up to 200 bp DNA
unreplicated at the 3’ end.
SOLUTION
Telomerase
- Specialized reverse transcriptase.
- Extends the end of the parental DNA by RNA-templated DNA synthesis.
- Responsible for the replication of the chromosome ends
- Conclusion: Telomerase extends the DNA, filling in the
gap due to the removal of the RNA primer
What is the structure of RNA?
RNA is usually single-stranded but it can fold into complex secondary structures called
hairpin-loops and stem-loops.
Loops = bases don’t match
Describe transcription steps
- Initiation does not require a primer: chain synthesis begins de novo
- Ribonucleotides are added to the 3’ - OH group of the growing RNA chain.
- DNA unwinds at the front of the transcription bubble and
- rewinds
Notes:
- SELECTIVE synthesis of RNA.
- Not all DNA is transcribed in a given cell.
- Synthesis is complementary and antiparallel to the
DNA template strand.
What is required for transcription?
1) DNA template
2) RNA nucleotides (rNTP’s)
3) RNA polymerase and other proteins.
RNA is transcribed from a DNA template. How? What direction read or synthesized?
- RNA is synthesized from one of the two DNA strands.
- Either DNA strand can be used as the template strand.
➢Template is always read in the 3’ -> 5’ direction.
➢RNA is synthesized in the 5’ -> 3’ direction.
Describe the transcriptional unit
- Region of DNA that codes for an RNA molecule and the sequences necessary for transcription.
- Three critical regions:
i. PROMOTER (upstream of start site, adjacent to gene)
ii. RNA CODING REGION (downstream of start site)
iii. TERMINATION SITE (downstream of start site) - Only the RNA coding region is transcribed.
What is a promoter again?
The PROMOTER is a DNA sequence that is recognized and bound by the transcription apparatus (RNA polymerase plus other proteins). The promoter
indicates the direction of transcription. Binding of the RNA polymerase to the promoter orients the enzyme towards the start site.
Describe prokaryotic transcription.
1.Initiation – assembly of transcription apparatus on the
promoter and begins synthesis of RNA
2.Elongation – DNA is threaded through RNA
polymerase, unwinds the DNA, adds new nucleotides to the 3’ end of the growing RNA strand
3.Termination – the recognition of the end of
transcription
Describe the prokaryotic RNA polymerase
- Many bacteria have multiple types of sigma factors, which help in the recognition of multiple classes of promoters.
- Without sigma, core enzyme initiates transcription randomly
- Holoenzyme is the complete enzyme complex composed of the core RNA polymerase and the sigma factor.
What do you know about the bacterial promoters and consensus sequences?
- Promoters contain short stretch of DNA that are conserved among promoters of different genes. These are called consensus sequences.
- Most common encountered sequences (or elements) are at -10 (Pribnow box) and -35 nucleotides upstream of the start site .
- Binding of transcription apparatus to these sequence orients the RNA polymerase towards the start site.
- 35 and -10 elements are not identical in all promoters.
- Each is a variation on a theme, i.e. consensus sequence.
- Variation affects the strength of the promoter. (strength = frequency of transcription)
- recA is a strong promoter.
Compare up and down mutations.
down mutations
- base substitutions that make the sequence less similar to the consensus sequences reduce the rate of transcription
up mutations
- sequence becomes more similar to the consensus sequences
How does prokaryotic transcription termination occur?
- Transcription ends after a terminator sequence is transcribed.
- Two major types of terminators in bacteria.
i. Rho-dependent (requires Rho protein)
ii. Rho-independent (also called intrinsic terminator)
Explain all that you know about Rho-dependent terminators.
1) Rho binds to RNA upstream of the terminator.
2) RNA polymerase pauses when it reaches the terminator sequence and Rho catches up.
3) Rho unwinds DNA-RNA hybrid using helicase
activity.
Describe the eukaryotic RNA polymerase II promoters. Compare the core promoter to the regulatory promoter.
CORE PROMOTER
- Extend upstream/downstream of transcription start site.
- Minimal sequence required for accurate transcription initiation.
- Includes a number of consensus sequences (common elements: TFIIB, TATA, Initiator and DCE) for transcription factor binding.
REGULATORY PROMOTER
- Located upstream of the core promoter, exact location can be variable.
- Transcriptional activator proteins bind to consensus
sequences and affect the rate of transcription.
Describe the assembly of the Basal Transcription Apparatus?
- TFIID binds to TATA box in core promoter
- preassembled holoenzyme consisting of RNA polymerase, transcription factors, and mediators all bind to TFIID
- transcription activator proteins bind to sequences in enhancers
- DNA loops out, allowing proteins bound to enhancer to interact with basal transcription apparatus
- activator proteins bind to sequences in regulatory promoter and interact with basal transcription apparatus through mediator
How does eukaryotic transcription termination occur?
- Transcription does not end at a specific sequence.
- Termination requires cleavage of the mRNA at a specific site.
- A 5’->3’ exonuclease degrades the remaining mRNA terminating transcription.
What are the three processes RNAi does?
- Condenses chromatin to supress transcription,
mRNA is not made. - The mRNA is destroyed before it is translated into protein.
- Inhibit translation, protein is not made.
Describe the origins of miRNA
- The precursors of miRNAs, called primary miRNA (pri-miRNA), are encoded by the genome. The relevant genomic regions are transcribed by RNA Pol II.
- In the nucleus, Pri-miRNA is cleaved by Drosha (RNAse III enzyme) into pre-miRNA, a stem-loop structure
- In the cytosol, Dicer cleaves the pre-miRNA into 19–25 nucleotide miRNA:miRNA duplex with no stem-loop
Compare siRNA and miRNA
siRNA
- Double-stranded RNA that contains up to 100 nucleotides
- Structure 21-23 nucleotide RNA duplex with 2 nucleotides 3‘ overhang
- Fully complementary to mRNA
- only one mRNA target
- Endonucleolytic cleavage of mRNA
miRNA
- Precursor miRNA (pre-miRNA) that contains 70-100 nucleotides with interspersed mismatches and hairpin structure
- 19-25 nucleotide RNA duplex usually with 2 nucleotides
3’overhang
- Partially complementary to mRNA, typically targeting the 3’ untranslated region of mRNA
- Multiple mRNA targets (could be over 100 at the same time)
- Translational repression.. Degradation of mRNA.. Endonucleolytic cleavage of mRNA (when there is a high level of complementary between miRNA and mRNA)
