ALL THE THINGS Flashcards
What is the central dogma of molecular biology?
DNA –> RNA –> Protein
What does a nucleotide consist of?
nitrogenous base, pentose sugar, 1 to 3 phosphates
What do nucleosides lack?
phosphates
What are the purines?
Adenine and guanine
How can you distinguish between the two purines?
Guanine has a carbonyl at C-6 and an amino group at C-2
Adenine has an amino group at C-6 only
What are the pyrimidines?
cytosine, thymine, uracil (RNA ONLY)
How can you distinguish between the pyrimidines?
Cytosine has a one carbonyl and one amino group attached to the ring
Thymine has 2 carbonyl groups and a methyl group attached to the ring
Uracil has 2 carbonyl groups attached to the ring
Structurally, how are purines and pyrimidines different?
Purines form a double ring structure, while pyrimidines are just one aromatic ring
When naming a nucleoside, what suffix is added to the nitrogenous base?
- sine for Purines(Ex. Adenosine)
- dine for Pyrimidines (Ex. Cytidine)
For a deoxyribose what prefix will you use?
deoxy-
How are the phosphate groups indicated when naming nucleotides?
5’ mono-, di-, tri- phosphates
What kind of bond is formed to join nucleotides together?
3’-5’ phosphodiester bond
What is the 3’ end called and what is the 5’ end of the phosphodiester bond called?
3’ hydroxyl
5’ phosphate
By what reaction is RNA degraded in a basic, alkaline solution?
hydrolysis; degraded to individual nucleotides (mix of 2’ and 3’ monophosphates)
The overall reaction is attack of the 3’-5’ phosphodiester bond that leads to full breakdown of the phosphate backbone
In a basic solution, is DNA degraded?
No, it is stable since it lacks the 2’ OH that is the target of hydrolysis
What are the 2 methylated bases seen in DNA?
5-methylcytosine and N6-methyladenosine (Methyl attached to the amino group at C-6)
Which methylated base is only present in Eukaryotes?
methylcytosine
What methylated bases are found in bacteria?
methylcytosine and methyladenosine
Which cytosine groups are methylated? What base must follow the cytosine in the sequence?
C’s followed by G’s
Only A’s part of what sequence can be methylated?
GATC
What are the two methylase enzymes that produce methylated bases?
Bacteria: DAM (deoxyadenosine methylase)
Eukaryotes: DNMT (DNA methyltransferase)
What is special about the recognition sites for methylase enzymes?
they are palindromic
Functions of methylation in Bacteria
control initiation of replication; discrimination of self DNA (methylated) from foreign DNA (non-methylated; discriminate old and new stands in mismatch repair; regulation of gene expression
Functions of methylation in Eukaryotes
regulation of chromatin structure and gene expression; can lead to gene splicing
Describe the orientation of DNA strands and explain why the sugar phosphate backbone faces outward while the bases are stacked in the interior
2 antiparallel strands
backbone is hydrophilic
bases are hydrophobic
Approx. how many base pairs per turn in a DNA double helix?
~10 base-pairs
How many H-bonds between A-T?
2
How may H-bonds between G-C?
3; 50% stronger, more difficult to dissociate
What are Chargaff’s Rules?
A = T ; C = G
A + G) = (C + T
Watson & Crick DNA is B-Form DNA. What are the other alternative structures?
A-DNA, Z-DNA, G-Quadruplex DNA
Describe G-Quadruplex DNA
has guanine tetrads (4 G’s in a plane connected by H-Bonding); stabilized by cation; stacks of 2 or more tetrads
Where is G-Quadruplex DNA found physiologically?
Telomeres
What kinds of grooves are present in DNA?
Major (wide) and Minor grooves (narrow)
What binds in the grooves?
DNA-binding proteins usually bind the major grooves
What are Inverted repeats?
Palindromic repeats; DNA sequences read the same 5’ - 3’ on complimentary strands
Is GACCAG a DNA palindrome?
NO!
5’-GACCAG-3
3’-CTGGTC-5
Is GACGTC a DNA palindrome?
YES!
5’-GACGTC-3’
3’-CTGCAG-5’
Palindromic DNA sequences are important for what?
they are important recognition sequences for restriction endonucleases and some transcription factors
Are palindromes interrupted in order to bind restriction enzymes?
NO
Are palindromes interrupted for transcription factors?
YES, by a few bases
Inverted repeates, when single-stranded, may form what?
hairpin or stem loop structures
Mirror repeats are important for the formation what kind of DNA?
triple stranded or Hoogsteen (H) DNA
What kind of mirror repeat forms H-DNA?
Repeat of a poly-puridine:polypyrimidine tract
The 3rd strand in triple-stranded DNA interacts with which groove? What type of base pairing is seen here?
Major groove
Hoogsteen base-pairing
Tandem (Direct) and inverted repeats are often involved in what?
structural chromosomal abnormalities
By what mechanism do tandem and inverted repeats cause abnormalities?
unequal crossing over in meiosis
What changes are associated with Tandem (direct) repeats?
deletions and duplications
Inverted repeats are associated with what changes?
inversions
Tandem (direct) repeats are involved in what kind of expansion seen in disorders like Huntingon’s?
trinucleotide repeat expansion
What is DNA denaturation?
H-bonds broken causing strand separation
How can DNA be denatured?
applying high heat or high pH
What is DNA renaturation?
separated strands can reassociate or reanneal, reforming H-bonds
How can the progress of denaturation and renaturation be followed?
by UV spectrometry
Does dsDNA (double stranded) absorb UV at 260 nm more strongly than ssDNA (single stranded)?
NO! ssDNA absorbs at 260 nm more strongly than dsDNA
When does UV absorbance increase?
during denaturation
What is the melting temperature (Tm) of DNA?
temperature at which dsDNA is 50% denatured
What is DNA hybridization?
annealing of a single strand of DNA to a complementary strand of a different DNA molecule
Is 100% complementarity required for hybridization?
NO! mismatches can be controlled by us in the lab
What is Deamination?
type of spontaneous DNA damage where the amino group on a nucleotide is lost
What are some examples of common DNA deaminations?
Cytosine’s amino group is changed to a carbonyl and forms Uracil
methylcytosine’s amino group is changed to a carbonyl and forms Thymine
What enhances DNA deamination reactions?
Nitrous acid (HNO2)
Methylcytosine is commonly deaminated to thymine. At what common sequences does this occur?
5’-CG-3’
MUTATION HOTSPOT
What is depurination?
spontaneous DNA damage that leads to the loss of the base from a nucleotide (usually a purine)
What is generated as a result of depurination?
apurinic sites (AP)
10,000 purines lost per cell, per 24 hrs
What are Mutagens and what kind of DNA damage do they cause?
physical, chemical, or biological agents that increase the rates of mutations; cause induced DNA damage
What happens when you expose DNA to alkylating agents?
Induced damage. Alkyl groups are added to bases. The result is mispairing between base-pairs
What bulky group commonly causes induced DNA damage?
Benzo(a)pyrene; oxidized in cells and binds covalently to guanine, distorting the double helix
What are intercalating agents?
cause induced damage; flat, planar molecules that slide between the stacked bases of the double helix; distort the double helix increasing base separation
What kinds of structural abnormalities are caused by intercalating agents?
insertions, deletions, and frameshift mutations
Name some common intercalating agents?
ethidium bromide, proflavin, acridine orange
What are the two causes of radiation-induced DNA damage?
X ray and UV exposure
What happens when DNA is exposed to X-rays?
free radicals generate that cause double stranded breaks
What happens when DNA is exposed to UV radiation?
pyrimidine dimers are generated (thymidine dimers); covalent bonds between adjacent pyrimidines in the same strand
What cause oxidative damage to DNA?
ROS (reactive oxygen species) like hydrogen peroxide, hydroxyl radicals, and superoxide radicals; generated by irradiation and from oxidative phosphorylation
How is DNA damaged from reactive oxygen species?
bases are oxidized; single or double stranded breaks occur
How is RNA different from DNA?
Ribose (not deoxy)
Uracil (not thymine)
Single stranded with dynamic secondary structure (complimentary to one DNA strand; Chargaff’s rules don’t apply)
Intramolecular base pairing can lead to partially double stranded structures (Watson-Crick or Hoogsteen pairing)
Unstable without protection from proteins
Unusual bases may occur (tRNA)
What are the unusual bases in RNA?
Inosine (deaminated adenine)
Pseudouridine (Uracil with a ribose attached)
7-methylguanosine (Guanine with a methyl group at C-7)
4-Thiouridine (Uracil has the oxygen at C-4 replaced with sulfur)
What structures are formed when inverted repeats are transcribed into RNA?
hairpin or stem loop
Complex RNA structures are generated by what?
H-bonds and base stacking
Are the 2D and 3D structures of RNA the same?
No. The 2D ones are used to clarify regions of bonding. 3D structures are very different
Describe the structure of tRNA
ssRNA with secondary structure; has 3 loops
