Section 2 - Module 6 Flashcards
What is the significance of 1952?
Year that Alfred Hershey and Martha Chase demonstrated that DNA and not proteins is transmitted on to progeny. DNA as the genetics material in Bacteriophages
Frederick Griffin’s work
Demonstrated cell can be transformed
What is the transforming principle?
DNA
What is Transformation?
acquiring new genetic material from uptake of external source
What bacteria did Hersey work with?
E/coli and their associated bacteriophage
Aaron Levene’s work?
Proposed tetranucleotide theory. Stating that DNA is made of repeating units called nucleotides
Albrecht Kossel’s work?
Determined nucleic acids contain four nitrogenous bases.
The four nitrogenous bases in DNA
Adenine (A), Cytosine (C), Guanine (G), Thymine (T)
The four nitrogenous bases of RNA
Adenine (A), Cytosine (C), Guanine (G), Uracil (U)
Significance of 1953?
Year Franklin and Wilkins devise the secondary structure of DNA. The double helix structure of DNA
DNA secondary structure
Structure of the set interactions between bases. Such as which parts of the strands are bound to each other and phosphate backbone.
Significance of 1948?
Year Erwin Chargaff discovered pattern in DNA base pairs. Nucleotide compositions DNA - A=T; G=C.
Describe nucleotide structure
Phosphate group, base (A, G, G, t), and deoxyribose sugar. Purines attached to pyridines
Purines
Adenine (A) and Guanine (G). They has an additional ring structure
Pyridines
Cytosine (C) and Thymine (T). They have an amide functional group
Phosphate group is attached to the __ carbon of deoxyribose sugar
5’
Base is attached to the _ carbon in deoxyribose
1’
What makes up deoxyribose sugars of DNA?
5 carbons with OH at 3’ carbon. Between 4’ and 1’ there is an ether (O).
What is different in RNA structure?
it is a ribose sugar and has an additional OH on carbon 2’. uses the U base T
Nucleoside
Sugar + Base (exposed nitrogenous bases)
Nucleotide
Sugar + Base + Phosphate group
Chargaff’s Rule
Purine(A+G)/Pyrimidines (T+C) = 1.0 approximately
Double stranded DNA
(C+T) = (A+G)
Rosalind Franklin and Maurice Wilkins
x-ray diffraction lead to discovery of DNA being a helix of constant diameter
Erwin Chargraff
Base pairing
Direction DNA spiraling around helix axis
anti-parallel (opposite directions)
Direction of reading DNA
5’ to 3’
What is perpendicular to the helix axis in DNA?
Base pairs
DNA two Grooves
Major and minor grooves allow for proteins to bind to and recognize DNA
Another way to refer to denaturing of DNA
Melting
What is “Melting”
Separation of two DNA strands, to single stranded DNA (ssDNA)
What is reversible posses in separation of DNA
Renaturation
Ways to denature or “melt” DNA
Increase temperature, reduce salt concentration, increase pH, solvents
Melting Temperature (Tm) of DNA
Defined as the temperature when DNA duplex is separated into single strands
What can DNA Tm indicate
duplex (hybridized DNA molecules) stability, higher Tm the more stable the DNA helix
How to measure DNA denaturation
absorbance. AS DNA duplex separated, the absorbance increases (hyperchromic shift).
What does higher Tm indicate?
More stable DNA helix
How does GC content (% G+C), effect melting temperature?
Higher GC content increases stability, therefore increasing melting temperature
How does salt concentration effect melting temperature?
Higher salt concentration, higher the melting temperature. Salt causes the phosphate backbone to the tightly packed, and this shield the negative charged of the phosphate backbone. Increases stability.
How can GC content in DNA classify organisms?
It is species specific
Does mutated of normal DNA have higher melting point?
Mutated melting rages are just different than “normal” ranges no specification of higher or lower
Molecular Biology Techniques involving DNA melting
polymerase chain reaction (PCR) and southern blotting
Equation for Melting Temperature
Tm = 81.5 + 16.6 lg [M] + 0.41(%GC) - 675/L
The three proposed models of DNA replication
Conservative, dispersive, and semiconservative replication
Correct model of DNA replication
semiconservative
What results of 1st round of Meselson experiment?
50% light and 50% heavy so NOT conservative replication
What is results of 2ndround of Meselson experiment?
There where some DNA that were 100% intermediate. So NOT intermediate replication
Semi-conservative replication
Each daughter cell consists of one parental strand and one newly synthesized strands based off a parental strand.
DNA synthesis Requirements
1) ssDNA template
2) all four dNTPs
3) DNA polymerase and other supporting enzymes
4) Free 3’-OH group
dNTPS
deoxynucleotide triphosphate, with each using a different DNA base: adenine (dATP), cytosine (dCTP), guanine (dGTP), and thymine (dTTP).
Mechanism of DNA Synthesis
Catalysis of phosphodiester bond between dNTPS and bases
Direction of DNA chain elongation
5’ to 3;
Template strand reading direction
3’ to 5’
Origin of Replication
the specific nucleotide sequence where replication begins
Where dies synthesis take place
Within a replication bubble
Where are DNA strands synthesized simultaneously
replication fork
What is a DNA molecule/region of DNA that replicates from a single origin of replication called?
Replicon
Replication is ______
Semicontinuous
Leading strand synthesis is _ and the direction of the fork
continuous
Lagging strand synthesis is _ and occurs in the opposite direction of the fork
discontinuous
Bacteria Genome
Circular
Bacteria Replication
Theta replication
Virus Genome
Circular
Virus Replication
Rolling circle replication
Eukaryotes Genome
Linear
Eukaryotic replication
Linear replication
Theta replication
1) single replicon (entire chromosome)
2) bidirectional replication w two forks within a single bubble
3) semi discontinuous in both replications forks
4) results in two circular DNA molecules
Rolling circle
1) no replication bubble
2) uncoupling of the replication of the two DNA molecules
3) replication is continuous
4) results in multiple circular DNA molecules
Linear
1) multiple replicons, origins or replication, and replication bubbles
2) bidirectional
3) Semi discontinuous at both replication forks
4) results in two linear DNA molecules
Four stages of replication
1) Initiation
2) unwinding
3) elongation
4) termination
Initiation
Initiator protein bind to the origin of replication (oriC) and a short section of DNA unwinds and proteins bein the ssDNA. ss-binding-protein keeps DNA separated and Helicases binds to the lagging strand template and breaks hydrogen bonds.
What bonds to helicases break
hydrogen bonds
Unwinding
Helicases break the hydrogen bonds between DNA strands while DNA gyrase (a topoisomerase) travels ahead of the fork and alleviates supercoiling caused by unwinding
What does unwinding cause without DNA gyrase to reverse
supercoiling
Chain Elongation
RNA primer (RNA nucleotides stretch) is synthesized by Primase. RNA primer provides a free 3’ OH for the DNA polymerases to use. RNA primer is replaced with nucleotides
Why do we require an RNA primer?
Because the production of RNA does NOT require a 3’ end
E. Coli principle replication enzyme
Pol III
DNA ligase
seals the nick in the sugar phosphate backbone
What DNA polymerase activity fills in the gap of DNA nucleotides (E. Coli)
Pol I
Exonucleases
Removes primers starting at the 5’ ends. Removes Newley incorporated nucletides that do not match the template strand
E. Coli polymerase that removes and replication RNA primers with DNA
pol I
How many E. Coli DNA polymerases are there
Five (Pol I to Pol V)
Eukaryotic DNA polymerases
Alpha, delta, and epsilon (all 5’ to 3’ activity) (delta and epsilon do 3’ to 3’ exonuclease activity)
Delta DNA polymerase
Lagging-strand synthesis of nuclear DNA, DNA repair, and translesion DNA synthesis
Alpha DNA polymerase
Initiation of nuclear DNA synthesis and DNA repair; has primase activity
Epsilon DNA polymerase
Leading-strand synthesis
Benefit of more origins in human DNA synthesis?
multiple origins ensure efficient genome replication in limited time
Eukaryotic DNA is packaged into ____
Chromatin
Telomeres
1) are the end of linear chromosomes
2) made up of G-rich short repeated sequences
3) stabilize chromosomes
specialized reverse transcriptase
4) extends the end of the parental DNA by RNA-templated DNA synthesis
5) responsible for the replication of the chromosomes ends
6) extends the DNA, filling int he gap due to the removal of the RNA primer
Transcription + translation =
gene expression
DNA replication
information transferred from one DNA molecule to another
Transcription
Information transferred from DNA to an RNA molecule
Translation
Information is transferred from RNA to a protein through a code that specify the amino acid sequence
Prokaryotes gene expression
transcription and translation both occur in the cytoplasm
Eukaryotic gene expression
Transcription in nuclear then pre-mRNA is processed to mRNA and leaves into the cytoplasm for translation
Protein coding RNA
mRNA
RNA unique to prokaryotes
CRISPR RNA (crRNA)
Folded complex of RNA called _
Hairpin-loops and stem-loops
Synthesis in respect to DNA template strand
complementary and antiparallel
Transcription
1) initiation does not require a primer
2) ribonucleotides are added to the 3’OH group of the growing RNA chain
3) DNA unwinds at the front of the transcription bubble
4)rewinds
Three requirements of transcription
1) DNA template
2) RNA nucleotides (rNTP’s)
3) RNA polymerase and other proteins
RNA transcription
Template is read in 3’ to 5’ direction, while RNA is synthesized in the 5’ to 3’ direction
ONLY the RNA coding region is transcribed
Promoter
upstream of the start site, adjacent to gene. Indicates the direction of transcription. Orients the enzyme towards the start site
RNA coding region
downstream of start site
Termination site
downstream of start site
Initiation (prokaryotic)
assembly of transcription apparatus on the promoter and begins synthesis of RNA
Elongation (prokaryotic)
DNA is threaded through RNA polymerase, unwinds the DNA, ass new nucleotides to the 3’ end of the growing RNA strand.
Termination (prokaryotic)
the recognition of the end of transcription
What do bacteria use to recognize promoters?
Sigma factors
what would happen without sigma?
core enzyme would initiate transcription randomly
Holoenzyme
in prokaryotes it is the complete enzyme complex composed of the core RNA polymerase and the sigma factor
Consensus sequences
short stretch of DNA that is conserved among promoters of different genes (prokaryotic thing)
Common sequences (or elements)
-10 (pribnow box) and -35 nucleotides which are upstream of the start site (+1). They are NOT identical in all promoters
Promoter sequences strength =
frequency of trasnciription
Strong promoter
recA
Down mutations
base substitutions that make the sequence less similar to the consensus sequences reduce the rate of transcription
up mutation
sequence become more similar to the consensus sequences
RNA transcription in prokaryotes
is initiated when core RNA polymerase binds to the promoter with the help of sigma
Terminators in bacteria
Rho-dependent (requires Rho protein) and Rho-independent (also called intrinsic terminator)
Rho-dependent termination
1) rho bind to RNA upstream of terminator
2) RNA polymerase pauses when it reaches terminator and Rho catches up
3) Rho unwinds DNA-RNA hybrid using helicase activity
Rho-independent termination
1) inverted repeats
2) polymerase pauses at Us
3) hairpin formation destabilize DNA-RNA hybrid
4) RNA transcript dissociated from RNA polymerase, DNA reanneals
Consensus sequences in order for eukaryotic transciption
TFIIB (-35), TATA(-25), initiator(+1), and DCE (+30)
Core promoter
extend upstream/downstream of transcription start site. Minimal sequence required for accurate transcription initiation. Includes a number of consensus sequences
Regulatory Promoter
located upstream of the core promoter, exact location can be variable. Transcriptional activator proteins binds to consensus sequences and affect the rate of transcription.
Order of Basal transcription apparatus assembly
TATA binding protein, general transcription factors, basal transcription apparatus
What is required for termination
cleavage of the mRNA at a specific site
What degrades the remaining mRNA terminating transcription?
5’ to 3’ exonuclease
