Genetic Information Transfer Flashcards
Genetic info is stored in a class of molecules called _____.
nucleic acids
The central dogma of genetic information transfer…
- DNA polymerase replicates DNA.
- RNA polymerase transcribes DNA > RNA.
- Ribosomes translate RNA to proteins.
Key structural difference beween RNA and DNA
- RiboNucleic Acid has an OH group on the 2’ carbon of its sugar.
- DeoxyriboNucleic Acid has a H on the 2’ carbon of its sugar (aka deoxidized).
Nucleotides are comprised of…
- a base (a purine or pyrimidine)
- a (deoxy) ribose, aka a sugar
- and phosphate PO4 (one or more)
Nucleotides are joined together through…
Nucleotides are joined together through phosphodiester bonds between the 3’ oxygen on each nucleotide’s sugar and the phosphate group of the neighboring nucleotide attached to its sugar’s 5’ carbon.
These phosphodiester bonds form the “backbone” of the polynucleotide.
Polynucleotides structures of one-strand and 2-strand DNA are stabilized through…
base stacking.
purine (A, G) and pyrimidine (T/U, C) pair via…
Hydrogen bonds.
The conformation of the base can affect H-bonding.
“sugar pucker” conformation…
The “sugar pucker” conformation affects nucleic acid’s tertiary structure by influencing whether it is tightly or loosely compacted.
It determines the distances between phosphates in the phosphodiester backbone.
The conformation of the base (syn vs. anti) determines how nucleotide bases interact.
DNA and RNA favor DIFFERENT sugar pucker conformations.
Z-form DNA includes both anti and syn conformations.
DNA structure basics
Typically 2-stranded in cells.
The two strands run antiparallel (3’ and 5’ on opposite ends).
Turns right-handed.
RNA structure basics
Typically single-stranded in cells.
Widely variable secondary structures.
Multi-functional molecule.
In forming nucleotide chains (polynucleotides), which end serves as nucleophile?
The 3’ hydroxyl is deprotonoated and acts as nucleophile, attacking the phophate off of the 5’ C on the next nucleotide.
Because of this, nucleotides are always added in a 5’ to 3’ manner.
nucleoside
A nucleoSIDE is a base connected to a pentose (5 carbon) sugar.
(no phosphate group)
glycosidic bond
The glycosidic bond links the base and the sugar to form a nucleoSIDE.
Occurs between the 1’ position carbon on the sugar and a nitrogen on the purine or pyrimidine base.
nucleoside monophosphate
A nucleotide with 1 phosphate group.
(phosphate-sugar-base)
This is what the final nucleic acid polymer is composed of.
nucleoside triphosphate
A nucleotide with 3 phosphate groups.
(phosphate-phosphate-phosphate-sugar-base)
These serve as high-energy building blocks used by the cells to synthesize the nucleic acid polymer.
Core chemical mechanism of joining nucleotides to form DNA:
1) Oxygen on 3’ carbon activated via deprotonation by a base.
2) 3’ O acts as nucleophile, attacking alpha phosphate of a nucleoside triphosphate.
3) Pyrophosphate (2 of the 3 phosphates) acts as a leaving group to drive the reaction forward.
The nucleotides always add to the 3’ end, so the strands are always extended from 5’ to 3’.
most common nucleotide base pairing pattern
H-bonds occur between the amino and carbonyl groups.
In Watson-Crick-Franklin base-pairing, H-bonds occur between the bases of two strands running anti-parallel to each other.
A-T (2-H bonds between adenine and thymine)
C-G (3 H-bonds between guanine and cytosine)
B-form double-stranded DNA helix
- Right-handed helix.
- Strands are antiparallel (maximizing WCF base-pairing).
- Sugar-phosphate backbone is oriented to the outside.
- Nucleobases are oriented to the inside.
Does B-form DNA need to unwind to interact?
NO! There is a wide, deep MAJOR groove
and a shallow, narrow MINOR groove in the B-form double helix.
Hoogsteen base pairs
When anti/syn-conformation nucleotides form base pairs.
(vs. traditional Watson-Crick-Franklin anti/anti base pairs)
Only the purine nucleotides (A, G) can take on syn conformation.
Linking Number
Linking number defines the topology of DNA twists.
LK=TW+Wr.
Linking number = sum of its twist and writhe.
Twist and writhe are inversely proportional.
B-form geometry, with equal numbers of Tw and Wr is very energetically favorable!
supercoiling
changing twists in DNA molecule.
(recall yoyo string example).
Supercoiled DNA is more compact.
If delta Lk=0, then DNA is relaxed.
If delta Lk<0, then DNA is unwound and negative supercoiling occurs. (most common in cells)
If delta Lk>0, then DNA is overwound and positive supercoiling will occur.
Topoisomerases
Topoisomerases are enzymes that change DNA topology by changing the linking number of DNA. This involves cutting, rearranging, and resealing the structure.
Help DNA compact and deal with disruptive structures that might form during replication and transcription.
Melting Temperature of DNA
Tmelting = the temperature at which the helix is half double-stranded and half-single-stranded.
(aka 50% denatured)
Stable helix = high Tm
Unstable helix = low Tm
Nucleosome
DNA wrapped around histone proteins.
The fundamental unit of eucharyothic genome packaging.
DNA must be unwrapped from around histone before a gene can be transcribed.
Chromatin
A form of DNA packaging. Tight and very compact, but not very accessible.
Histone acetylation
Marker that makes DNA MORE ACCESSIBLE. Found in euchromatic regions.
Histone methylation
Marker making DNA INACCESSIBLE. Found in heterochromatic regions. Also marked by binding of heterochromatin proteins.
What is the preferred sugar-pucker conformation of DNA nucleotides?
C2 endo conformation; dominates B-strand DNA.
C3 endo conformation can be adapted by A-strand DNA.
phosphodiester bond
phosphodiester bond occurs between the 3’ OH and the phospho group on the 5’ C. Links the nucleotides in the polynucleotide strand.
A base activates the OH for nucleophilic attack of the dNTP (deoxyribonucleotide 5’-triphosphates). diphosphates act as leaving group.
Why is DNA the principal storage, and not RNA?
RNA is more suseptible to degradation than DNA. The 2’ OH makes it more reactive and can break down the chain.
DNA Polymerase
the enzyme (and protein) that catalyzes nucleotide addition for replication.
Requires a primer–a short double-stranded segment–to start replication.
Uses metal ions (Mg2+) to interact with triphosphate groups of incoming nucleotides, stabilizing neg charged transition state.
Why does DNA polymerase require a primer?
base-pairing provides a portion of the stability; the primer facilitates base STACKING, which is energetically favorable.
exonuclease
When DNA polymerase makes mistakes and its own proofreading doesn’t catch it, it can cause instability that leads the DNA strand to the exonuclease site on the polymerase.
The exonuclease removes the erroneous base, and polymerase has another chance to add the correct base. Cleaves 1 nucleotide at a time from the END of a strand.
frequency of DNA mistakes
DNA polymerase error rate is about every 1 in 100,000 to 10,000,000 additions
What happens at the DNA replication fork?
leading strand and lagging strand split in opposite directions.
The leading strand undergoes continuous synthesis 5’ to 3’.
The lagging strand requires a series of primers to synthesize 5’ to 3’. Therefore, it is made of a series of discontinuous Okazaki fragments, linked together by DNA ligase.
Telomerase
prevents shortening at the end of the lagging strand during DNA replication.
helicases
provide the energy to unwind double-strand DNA
primase
adds DNA primers for replication.
Pol III and Pol I
Two types of polymerase used in DNA genome replication.
Pol III is the workhorse, built for speed.
Pol I is the craftsman, built for accuracy.
General steps in DNA replication
- helicases unwind the double strand
- primase adds primers
- Pol III starts replicating
- unwinding continues
- lagging strand needs more activity–replace RNA starters with DNA and link fragments with ligase.
The product is semi-conservative replication!
5 basic steps to DNA repair
- detection
- incision
- excision
- synthesis
- ligation (ligase seals)
2 main paths to mutation in the genome:
- polymerase makes a mistake and misses it proofreading.
2. chemistry takes a toll; modified base will lead to incorrect base pairing during future replication.
The lac operon
model from E. coli using transcriptional activators and repressors to regulate gene expression.
DNA binding proteins in gene regulation and expression
transcriptional activators and repressors make sequence-specific interactions with DNA.
Sequences are read through side-chains that H-bond with DNA bases. Does NOT require melting the DNA!
DNA binding activity is often separate from regulatory activity (proteins are modular).
regulation of chromatin structure (a type of epigenetic regulation)
relation of chromatin structure by histone proteins; positioning and post-translational modifications
chemical modification of nucleosides (a type of epignenetic regulation)
DNA base is modified
heterochromatin regions of DNA…
are transcriptionally silent
aka not accessible.
euchromatic regions of DNA…
are transcriptionally active
aka accessible
heterochromatic regions of chromatin…
are transcriptionally silent
aka not accessible.
euchromatic regions of chromatin…
are transcriptionally active
aka accessible
acetylation (lysine)
makes DNA more accessible.
Tends to be found in euchromatic regions.
Active gene expression site.
acetylation (lysine)
makes DNA more accessible.
Tends to be found in euchromatic regions.
Active gene expression site.
Provides binding sites for transcriptional activators.
5-methylcytosine acts to
silence gene expression
5-methylcytosine acts to…
silence gene expression
DNA repair pathways–direct repair of alkylation by methyltransferase
- Carbonyl oxygen of base (G) bonds to an alkyl chain of some sort.
- AGT protein bonds with alkyl via SN2, base is L.G.
- 1 AGT protein sacrificed for each alkylation!
DNA repair pathways–mismatch repair of newly-synthesized strand
- newly-synthesized daughter strand has a mismatch, parent strand still methyl-marked.
- proteins assembled to recognize and spool around the mismatch; cleave daughter strand at -CH3.
- exonuclease “chews” back fragment on daughter strand to the preceding -CH3 so polymerase can try again.
DNA repair pathways-base excision repair
- Deamination leaves DNA with wrong base OR depurination leaves DNA with NO base.
- glycosylase enzyme cleaves the base
- AP nuclease recognizes the empty space, cleaves backbone
- DNA polymerase extends chain with new base
- ligase “links” with a new phosphodiester bond
DNA repair pathways-nucleotide excision repair
- pathway for chemical modifications to DNA that disrupt base-pair interactions–deamination, alkylation, depurination or UV-induced.
- NT excision surveillance complex recognizes, attaches, and cleaves 1 strand on either side of the dimers, leaving a knicked substrate.
- helicase separates the strands, removing dimer fragment.
- DNA polymerase extends chain at the gap.
- Ligase seals.
UV dimers
UV-induced dimerization are molecular “lesions” formed on stacked pyrimidine bases (T or C); sometimes many nucleotides long
deamination
nucleotide in sequence has NO BASE. Fixed via base-excision repair.
depurination
removal of a purine base. Fixed via base-excision repair.
endonuclease
An enzyme that cleaves the phosphodiester bond WITHIN a chain of nucleotides (vs. exonuclease cleaving from the end).
Homologous Recombination
- For big repair jobs.
- Repairs DNA double strand breaks via Holliday Junction.
- find homologous strand, close match
- chew back strands to have 3’ overhang to build off of.
- strand invasion is when 3’ end interacts with the in-tact chromosome; forms 4-way Holliday Junction
- resolved by cutting junction either vertically or horizontally–2 diff outcomes!
- this is also a tool of the genome to generate variety
Sequence-specific recombination –Tyrosine recombinase
- For big repair/ rearrangement.
- Begins with Tyrosine OH attacking phosphate in DNA backbone
- 2 sequential single-strand breaks
- holliday junction intermediate
Sequence-specific recombination – Serine recombinase
- For big repair/rearrangement
- Both strands cleaved simultaneously; rotations; ligase seals
- NO holliday junction
Recombination – Transposons
- transposons = mobile DNA elements that move around the genome
- developed by Barbara McClintock via studying corn pigmentation
- transposons give a mosaic blend of different genotypes
- 2 types:
1) Retro-transposons, “copy/paste” via RNA intermediate
2) DNA transposons, “cut/paste” (no RNA intermediate)
RNA polymerases
- catalyze RNA synthesis from DNA templates (for eventual translation into proteins)
- Don’t require a primer (like DNA does)
- Chemistry is energetically favorable, so can unwind as it goes (without needing helicase to open like DNA does)
- only one strand transcribed at a time (results in a copy of the “coding strand” using the “template strand”).
- No exonuclease site, but can still back-track to make corrections
promoter sequences
- specifies where RNA polymerase will initiate transcription
- RNA polymerase binds to promoter
- sigma subunit tells RNA polymerase what sequences are promoters
- strands melt and only one strand of DNA can be transcribed at a time.
Terminating transcription
in prokaryotes, there are 2 primary mechanisms to STOP transcription.
1) Rho dependent
2) Rho independent
They both disrupt RNA polymerase from interacting with template strand.
Rho-dependent transcription termination
relies on the helicase (unwinding)
Hairpin (Rho-independent) transcription termination
- relies on a G/C rich region to make a hairpin structure, with U-rich region downstream
- intrinsic to the sequence
epigenetics
changes in gene expression not caused by changes in DNA sequence
3 types for this class:
1) regulation of chromatin structure
2) chemical modification of nucleosides
3) microRNA-mediated regulation
DNA wraps around ____ proteins to form _____. Then further compacted to _____, then chromosomes.
DNA wraps around histone proteins to form nucleosomes. Then further compacted to chromatin, then chromosomes.
3 major classes of RNA in the cell
1) messenger RNA (mRNA)–encodes protein sequences
2) transfer RNA (tRNA)–
3) ribosomal RNA–
introns vs. exons
Introns - gene sequences removed from the RNA transcript
Exons - sequences that remain in the RNA transcript; EXONS are EXPRESSED
micro RNAs
another type of RNA molecule that regulates gene expression
- small fragments encoded in the genome
- use base-pairing interactions to target mRNAs for regulation
spliceosome
- in Eukaryotes, the spliceosome splices/removes introns from RNA
- happens in the nucleus
Processing of mRNA before exiting nucleus (in Eukaryotes)
To signal the mRNA is mature:
1) splicing of introns
2) 5’ cap (7-methylguanosine) put on mRNA
3) 3’ poly A tail added on by Polyadenylate Polymerase (PAP)
Stabilizes and prevents foreign invaders
tRNA
tRNAs are adaptor molecules that link a triple codon to its associated amino acid.
- Use Watson-Crick-Franklin base-pairing interactions to read triplet codons in the mRNA
- “charged” with their amino acid by tRNA synthetases
genetic code for translation
A table mapping codons (each made of 3 nucleotides) to specific amino acids.
Redundant and universal.
Wobble hypothesis
- ~45 tRNA synthetases, but 64 codons
- third base of codon is more relaxed and gives coding flexibility; the same synthetases can read multiple codons
- Inosine (I) can be put in 3rd spot and can pair with multiple nucleobases
start codon sequence, Shine-Dalgarno sequence
start codon sequence: AUG
The Shine-Dalgarno sequence indicates to the ribosome which AUG codon to start at.
stop codon sequences
UAG, UAA, UGA
“Release factor” protein in ribosome recognizes the stop codon on AA and promotes polypeptide release and translation termination.
Nucleotides that STOP synthesis lack a 3’ OH!
3 consequences of mutations, defined by genetic code and reading frame
1) Silent mutation - no affect on polypeptide
2) Mis-sense mutation- changes mRNA sequence from a codon for one amino acid to a codon for another; may not have major affect
3) Non-sense mutation - change the mRNA sequence form a codon for an amino acid to a STOP codon, terminating translation; can cause BIG problems
The ribosome
- The site of protein synthesis and translation.
- Has both RNA and protein components (similar to splicisome)
- Responsible for:
1. initiating synthesis at start codon AUG
2. synthesis of peptide bonds between amino acids
3. terminating synthesis at stop codon (UAG, UAA, UGA)
Polymerase Chain Reaction (PCR) technique for making synthetic DNA
If we know the end (primer) of a DNA segment of interest, we can massively amplify it via repeated cycles of denaturing, primer annealing, and polymerase extension. The primer is incorporated into the product sequence.
Overlapping DNA fragments can be assembled into larger genes via Gibson assembly (like a recombination reaction)
plasmid
a circular DNA molecule that replicates separately from the host molecule.
oligonucleotide (oligos)
short DNA sequence that can be synthesized chemically for use of changing the genetic composition of cells.
Sanger Method of reading DNA sequences
Exploits nucleotide structure; remove 3’ hydroxyl (di-deoxynucleotide) from one of the nucleotides (ATC or G).
Put a small amount of this in, and every so often (1 in 50 times or so); strand will terminate wherever it is incorporated. By looking at the intermediate products, you know the position of that sequence.
Repeat for others (ATC or G), and you can run on gel and read the entire sequence!
In modern times, fluorescently labeled ddNTPs (one color for each reaction) so you can do this in a single reaction.
Nanopore method of DNA sequence reading
DNA fed through a protein-based pore in a membrane. Changes in conductance as the DNA goes through the membrane distinguishes the slightly different voltage between A, T, C, & G.
Less accurate, but can read millions of bases in a single read.
self-splicing introns
segments of RNA that can splice themselves out of an RNA polymer without the assistance of any proteins.
2 types of self-splicing introns
- Group I Introns –Use a free guanosine nucleotide to initiate cleavage.
3’ to 5’ nucleophilic attach on phosphodiester backbone joins exon ends. Released intron is a linear fragment. - Group II Introns –Use an adenosine within the intron to initiate cleavage.3’ to 5’ nucleophilic attach on phosphodiester backbone joins exon ends. Released intron is a lariat structure (loop-like).
2 types of self-splicing introns
- Group I Introns –Use a free guanosine nucleotide to initiate cleavage.
3’ to 5’ nucleophilic attach on phosphodiester backbone joins exon ends. Released intron is a linear fragment. - Group II Introns –Use an adenosine within the intron to initiate cleavage.3’ to 5’ nucleophilic attach on phosphodiester backbone joins exon ends. Released intron is a lariat structure (loop-like). Splicisome might be evolutionarily related to the group II intron.
