Exam 1 Flashcards
What did Rosalind Franklin discover?
Using X-ray diffraction on DNA the double helix structure was discovered
Structure of DNA
Each nucleotide is composed of a sugar, a phosphate, and a base (guanine, cytosine, adenine, thymine or uracil).
Nucleotides are linked by covalent phosphodiester bond between sugar and phosphate
(sugar–phosphate backbone)
DNA: two polynucleotide chains held together by hydrogen bonds between the paired bases.
(A = T) (G ≡ C)
Run antiparallel (opposite chemical polarities: 5’ of sugar and 3’ of sugar give rise to polarity)
Human genome
full set of human chromosomes
Human karyotype
ordered display of full set of 46 chromosomes
Which DNA sequence elements are necessary for the duplication of a chromosome and then segregation at mitosis?
Replication origins
Centromere
2 telomeres (marking ends of each chromosome)
Overview of the cell cycle
Interphase: DNA replication begins at replication origin giving duplicated chromosomes
M phase: centromeres attach duplicated chromosomes to mitotic spindle = one copy distributed to each daughter cell
What is the nucleolus
a well-organized region in the interphase nucleus where parts of different chromosomes that carry genes that encode ribosomal RNAs come together
What is the function of the nucleolus
rRNAs are synthesized + combine with proteins to form ribosomes
Structure of the interphase nucleus
Interphase chromatin:
Heterochromatin: contains few genes that don’t usually get expressed due to its highly condensed form
Concentrated around centromeres + telomeres
Appear as dense regions of chromatin
Euchromatin: actively transcribed + not as condensed
Nucleolus contains the genes for rRNAs
What is the nucleosome
first and most fundamental structural unit of chromatin packing
convert DNA molecules in interphase nucleus into a chromatin fiber (clusters of closely packed nucleosomes)
Features of experimentally unpacked chromatin fiber
looks like beads on a string
string - the DNA
bead - a nucleosome core particle (DNA wound around a core of histone proteins)
Exposed DNA between core particles - linker DNA
Process of experimentally unpacking chromatin fiber
(structure of chromatin fiber / nucleosome core)
Nucleosome core particle can be released from chromatin by digestion of the linker DNA with a nuclease, which cleaves the exposed linker DNA but not the DNA wound tightly around the nucleosome core.
When the DNA around each isolated nucleosome core particle is released, its length is found to be 147 nucleotide pairs; this DNA wraps around the histone octamer (2 sets of 2 histones)
Levels that give rise to highly condensed mitotic chromosome
short region of DNA doube helix > beads on a string chromatin fiber > further nucleosome packaging via H1 (pulls adjacent nucleosomes together) > chromatin fiber folded into loops via nonhistone chromosomal proteins = interphase chromosome > more packing to form mitotic chromosome
How do chromatin-remodeling complexes work?
Reposition the DNA wrapped around nucleosomes
Use energy from ATP hydrolysis to loosen the nucleosomal DNA + push it along the histone octamer
Either exposing or hiding a sequence of DNA controlling its availability to other DNA-binding proteins
What aids changes in nucleosome structure to allow access to DNA
chromatin-remodeling complexes
histone-modifying enzymes
How do histone-modifying enzymes work?
All histones in the core have tails that extend from each nucleosome core particle
Each histone can be modified by the covalent attachment of diff groups to the tails (e.g addition/removal of acetyl, phosphate, methyl etc)
These modifications generally serve as docking sites on histone tails for nonhistone chromosomal proteins which then have different functional outcomes (e.g gene silencing, gene expression, heterochromatin formation, etc)
How does heterochromatin form and spread?
heterochromatin-specific histone tail modifications attract heterochromatin-specific proteins that reproduce the same histone modification on neighbouring nucleosomes
Heterochromatin spreads until it encounters a barrier DNA sequence that blocks further propagation into regions of euchromatin
Study that demonstrated DNA is the genetic material
Avery, MacLeod, McCarty
Prepared an extract from S strain + fractioned it into classes of molecules (RNA, protein, DNA, lipid, carbohydrate)
Tested the molecules ability to transform R-strain cells
Only the DNA was able to transform R into S strain
Concluded: Molecule that carries heritable ‘transforming principle’ is DNA
Outline of study regarding pathogenic and harmless bacteria
Study by Griffith:
S strain of S.pneumoniae = disease causing
R strain = harmless strain
Injected S strain = mouse dies of infection
Injected R strain = mouse lives
Injected heat-killed S strain = mouse lives
Injected mix of heat-killed S and live R = mouse dies of infection + living S strain is recovered
X-chromosome inactivation
males (with one X chromosome) females (with two X chromosomes but one inactivated)
Females have two X chromosomes (one from the mother and one from the father).
Early in development, randomly one of these X chromosomes becomes inactivated and condensed into heterochromatin.
Once an X chromosome is inactivated in a cell, that same X stays inactivated in all cells that come from that original cell.
The inactivated X chromosome is seen as a Barr body (a small mass of chromatin in the nucleus).
What is the biological significance of X-chromosome inactivation in females?
dosage compensation: Men and women now have similar levels of X-chromosome-related gene expression
Consequences of incomplete x-chromosome inactivation in females?
more expression of certain genes, especially at the tip of the chromosomes. Tip of chromosomes contain certain genes of the immune system, this is one of the ideas why females are better in combatting infections diseases such as Covid 19. The price they pay is: more autoimmune diseases (about 4x more) in women.
Genes at the tip of X, that are not silenced, even in female cells with inactivation of one X chromosome.
= female cells have the capacity to make more IFNs = protect them against developing severe Covid-19 symptoms
What enables a cell to replicate its genes
The ability of each strand of DNA to act as a template for producing a complementary strand
What is the first step of DNA replication
Parent DNA separation
- initiator proteins (helicase) bind to replication origins
- locally pull apart the two strands of the double helix (breaking the hydrogen bonds between base pairs)
Form replication forks at each replication origin
What is proofreading in DNA replication?
Done by DNA polymerase (has a P site for polymerization activity and E site for proofreading/editing)
If incorrect base pair then it clips off the mispaired nucleotide and tries again
Replication forks at a replication origin
- two replication forks form at a replication origin
- move away in opposite directions (bidirectional)
- movement driven by the replication machine (opening up 2 strands + copying DNA) which involves helicase, DNA polymerase, and single-strand DNA-binding proteins (prevents strands from reforming + keeps them elongated)
Which parent strand is the lagging and leading strand? and in which direction is new DNA synthesized?
New DNA is synthesized in a 5’ to 3’ end
Remember strands run antiparallel
Leading strand:
continuous replication as DNA is synthesized towards helicase
Lagging strand: noncontinuous replication, okazaki fragments, moves back along the template strand towards the fork to synthesize the next fragment
How is replication started
Primase synthesizes the RNA primer which makes a short length of RNA giving a 3’ starting point
For leading RNA primer is only needed to start replication @ replication origin
For lagging new primers repeatedly needed
What is the role of the sliding clamp in DNA replication
keeps DNA polymerase attached to the template, allowing it to move along without falling off
On lagging: clamp detatches each time the polymerase completes an okazaki fragment and reattaches each time to start a new one
What is the role of the clamp loader in DNA replication
uses energy of ATM hydrolysis to lock the sliding clamp onto DNA
How are replication proteins arranged when a replication fork is moving
Lagginf strand has been folded to bring its DNA polymerase in contact with the leading strand DNA polymerase + bring the 3’ end of each completed okazaki fragment close to the start site
What is the role of the DNA ligase in DNA replication
uses energy of ATP hydrolysis to join okazaki fragments
What is the role of DNA topoisomerase in DNA replication
produces transient nicks in the DNA backbone to relieve the tension built up by unwinding the DNA ahead of helicase
How are okazaki fragments joined together
nuclease degrades RNA primer > DNA polymerase replaces RNA primer with DNA > DNA ligase joints the 5’ phosphate end of a DNA fragment to adjacent 3’ hydroxyl end of the next
How is DNA replication finished off and what are the issues encountered?
Leading strand is replicated all the way to the chromosome tip
Lagging strand ends cannot be completed because once the final RNA primer has been removed there is no mechanism for replacing it with DNA (chromosome ends would keep shrinking with each cell division)
How is the finishing up dilemma of the lagging strand dealt with?
Template strand is extended beyond the DNA that is to be copied via telomerase (adds to the telomere repeat sequences at the 3ʹ end of the template strand) which then allows the newly synthesized lagging strand to be lengthened by DNA polymerase
Telomerase carries its own short piece of RNA template that is complementary to the DNA repeat sequence
What happens when damage occurs to DNA during replication
+ examples
Chemical modifications of nucleotides, if left unrepaired, produce mutations.
Deamination of cytosine produces uracil.
During replication base pairs the U with A (when it should be G and C which correctly forms on the other template strand)
Depurination, if uncorrected, can lead to the loss of a nucleotide pair.
When the replication machinery encounters a missing purine on the template strand, it can skip to the next complete nucleotide
Producing a daughter DNA molecule that is missing one nucleotide pair
Cancer incidence with age
Because cells are continually experiencing accidental changes to their DNA—which accumulate and are passed on to progeny cells when the mutated cells divide—the chance that a cell will become cancerous increases greatly with age.
How does mismatch repair work
Mismatch repair eliminates replication errors and restores the original DNA sequence.
Repair machinery must replace the incorrect nucleotide on the newly synthesized strand, using the original parent strand as its template.
Eliminates the error, and allows the original sequence to be copied during subsequent rounds of replication.
Difference between RNA and DNA
RNA contains the sugar ribose
DNA has deoxyribose sugar
RNA contains the base uracil, which differs from thymine
RNA is single stranded
Process of transcription
Opening small portion of DNA to expose bases
One strand serves as the template
RNA transcript produces RNA chain 5’ to 3’ direction
RNA polymerase covalently links incoming ribosenucleoside triphosphates to growing RNA chain, and unwinds DNA helix ahead, and catalyses formation of phosphodiester bond between nucleotides
DNA strands in transcription
Transcription of a gene produces an RNA complementary to one strand of DNA
The template strand (DNA) is used to guide the synthesis of the RNA molecule.
The nontemplate strand (DNA) called the coding strand because its sequence is equivalent to the RNA product
Which DNA strand serves as the template varies
What signals bacterial RNA polymerase where to start and stop
Bacterial RNA polymerase contains a subunit called sigma factor that recognizes the promoter of a gene
Once transcription starts, sigma factor is released, + polymerase moves forward, synthesizing the RNA
Elongation continues until the polymerase encounters a sequence in the gene called the terminator
After transcribing this sequence into RNA, enzyme halts + releases both the DNA template and newly made RNA
Polymerase then reassociates with a free sigma factor and searches for another promoter to begin the process again
What determines which DNA strand is transcribed
The polarity of the promoter orients the polymerase
How is transcription initiated in eukaryotes?
Eukaryotic RNA polymerase II requires a set of general transcription factors
Eukaryotic promoters contain a DNA sequence called the TATA box which is recognized by a subunit of the general transcription factor TFIID, called the TATA-binding protein (TBP)
The binding of TFIID enables the adjacent binding of TFIIB.
The rest of the general transcription factors, as well as the RNA polymerase itself, then assemble at the promoter.
TFIIH pries apart the double helix at the transcription start point, using the energy of ATP hydrolysis = exposes the template strand of the gene
TFIIH phosphorylates RNA polymerase II, releasing the polymerase from most of the general transcription factors, so it can begin transcription
Once the polymerase moves away from the promoter, most of the general transcription factors are released from the DNA; the exception is TFIID, which remains bound through multiple rounds of transcription initiation
What are exons and introns
exons: coding sequences/regions on a gene that become expressed
introns: noncoding sequences that interrupt exons
What is RNA splicing
introns are removed from freshly synthesized RNA and exons are stitched together
What is alternative splicing
Some pre-mRNAs undergo alternative RNA splicing to produce different mRNAs and proteins from the same gene
Exons can be skipped over by the spliceosome
Skipping occurs when the splicing signals at the 5ʹ end of one intron are paired up with the branch-point and 3ʹ end of a different intron.
Order of exons cannot be rearranged
How is splicing carried out
via snRNPs (U1, U2, and U6)
U1 recognizes the 5’ slice site and U2 recognizes the lariat banch-point site through complementary base-pairing
U6 rechecks the the 5’ splice site by displacing U1 + base-pairing with this intron sequence
Formation of spliceosome active site
Splicing reactions occur
Spliceosome deposits exon junction complex on the mRNA to mark splice site as successfully completed
