DNA replication Flashcards
B-DNA
- “normal” DNA
- Right handed
- Pitch: 35 A (10.4 bp)
A-DNA
- Right handed
- shorter/wider than B-DNA
- Pitch: 25.3 A
Z-DNA
- Left handed
- Tall, narrow
- Pitch: 45.6 A
Supercoiling
- DNA in the cell must be organized
- Allows for packing of large DNA molecules within the cells
- Allows for access of proteins to read the information in DNA sequence
- Many circular DNA are supercoiled
L
- linking number
- number of times strands go around each other; number of times plane is crossed
T
- twist
- number of turns of double helix
W
- writhe
- number of times the double helix wraps around itself
L,T,W association in closed system
- L=T+W
Relaxed DNA
- W =0
- L=T=# of bp divided by 10.4
(negatively) supercoiled DNA
- W ≠ 0 ⇒ compacted DNA
How to change linking # of circular or closed DNA
- Cut, twist, reattach
Function of topoisomerases
- change the linking number
Type I topoisomerases
- Add negative supercoils
- Cleave 1 strand
Type II topoisomerases
- Remove negative supercoils
- Cleave 2 strands
OriC
- In E coli: one origin of replication/chromosome
- 245 bp
- highly conserved sequence elements
- Binding site for initiator protein: DnaA
ARS
- In eukaryotes
- Approx. 400 well defined origins
- Entire genome replicated 1X/cycle
- Regulation due to cyclin proteins and cyclin-dependent kinases (CDKs)
- Cyclins are ubiquinated for proteolytic destruction at the end of the M (mitosis) phase
OriC and ARS similarities
- A,T rich
- easy to “melt” DNA into single strands
Helicase
- Melts double stranded DNA
- Use ATP
Primase
- Make RNA primers
- RNA primers are added first to replicating strand; later removed
DNA polymerase I
- Replaces RNA primer with DNA
- 5’ to 3’ exonuclease activity
- Not ideal for replication
- Slow, low processivity
DNA polymerase III
- Principal replication polymerase
Ligase
- Seals “knicks”
- Puts Okazaki fragments together
Telomerase
- replicates telomeres
Topoisomerase I
- Behind replication fork (on 2 daughter DNA)
Topoisomerase II
- Ahead of replication fork (on parent DNA)
Leading strand synthesis
- Replicated continuously
- DNA polymerase 𝛅
- DNA polym III in prokaryotes
Lagging strand synthesis (what and proteins)
- Generated in small steps
- Okazaki fragments
- Priamse: makes 15 bp RNA primer for each okazaki fragment
- DNA polym. III
- Does all leading strand synthesis
- Makes DNA from from 1 primer to the next on lagging strand
- DNA polym. I
- Replaces RNA in primer with DNA
- Ligase
- Seals knicks
- Single stranded binding proteins
- Stabilize single stranded DNA
- Prevent strands from sticking back together
Subunits
- 20 individual peptides combined
- In pol III: 10 types of subunits
Homotetramer
- 4 identical subunits
- “Same” “four”
Heteropentamer
- 2 or more different monomers
- “Different” “five”
Holoenzyme
- does most DNA synthesis activity
- proofreading capabilities that correct replication mistakes by means of exonuclease activity working 3’→5’
- high processivity (i.e. the number of nucleotides added per binding event)
Central structure
- hold 2 holoenzymes together and allows them to move together
a subunit
- catalytic subunit
- Has a groove for DNA to slide along and active site where nucleotides are added
e subunit
- proofreading subunit
- Just behind a; removes incorrect nucleotides
b subunit
- processivity subunit/beta-clamp
- Forms “donut” around DNA to prevent pol III from falling off
What is crossing over?
- The exchange of genetic material between homologous chromosomes that results in recombinant chromosomes during sexual reproduction
When does crossing over occur?
- Prophase I (meiosis)
Quartenary stucture of pre-recombinase
- homotetramer
How does Cre recombinase work?
- catalyzes site specific recombination between 2 DNA recognition sites
- cyclic recombination
- @ LoxP sites (8 bp sequence with 34 bp palindromic sequences on either side)
What do tyrosine resiudes do in cre recombinase?
- nucleophilic attack on phosphate bonds of DNA
How is cre recombinase similar to topoisomerase I?
- forms covalent bonds between hydrogen bonds and tyrosine residues (how it controls strand breaks and combine DNA)
What is a mutation?
- alteration in DNA structure that produce permanent changes in the genetic information encoded therein
When do mutations take place?
- during cell division (replication)
- environmental factors
- UV
- chemical
- viruses
DNA damage vs mutation
- mutation: affects nucleotide sequence (can’t be fixed)
- Damage: damage to bonds, etc; abnormalities in DNA
- can be fixed
3 causes/types of DNA damage and the mutations they cause
- UV light
- pyrimidine dimers
- Chemical damage
- point mutation
- Intercalating agents (insertion of molecule between DNA planes)
- frameshift mutation
3 steps of DNA repair
- Abnormal DNA is recognized
- abnormal DNA is removed (upstream and downstream)
- normal DNA is synthesized
3 enzymes of DNA repair
- polymerase III
- Ligase
What is an Ames test?
- Indicates mutagenic potential of a compound
- Add compound to plate of salmonella
- see if it grows in His free medium
- Colonies (+test) indicates the compound mutated the salmonella, restored ability to synthesize His
Use of liver cells in Ames test
- Can treat the compound with liver cells to turn it into a mutagen, to see if the body processes it into a mutagen
RNA polymerase: quartenary structure in prokaryotes
- 5 subunits (pentamer)
- a: (2) assembly and binding to UP (upstream promoter) elements
- B: main catalytic subunit
- B’: responsible for DNA binding
- o: directs enzyme to promoter
- w: protect polymerase from denaturation
What does RNA polymerase I transcribe in eukaryotes?
- pre rRNA (turns into rRNA)
What does RNA polymerase II transcribe in eukaryotes?
- pre mRNA
- some snRNAs
What does RNA polymerase III transcribe in eukaryotes?
- pre tRNAs
- 5S rRNA
- some snRNAs
Initiation
RNA polymerase binds to promoter on DNA (by transcription factors in eukaryotes)
Elongation
- Elongation factors enhance activity of RNA polymerase
- RNA polymerase reads template strand
- Goes in 3’ to 5’ direction
Termination
- In bacteria: stops due to hairpin loop
- In eukaryotes:cleavage of the new transcript followed by template-independent addition of adenines at its new 3’ end, in a process called polyadenylation
- Pol II released
Template strand
- Non-coding
- template for RNA polymerase
- Antisense
Coding strand
- Non-template strand
- has same sequence as RNA transcript
- Sense
Promoter
region of DNA that initiates transcription of a particular gene
+1
- 1 bp away from promoter
- On coding strand
Why different sigma subunits in prokaryotes
- bind different promoters
- transcribe unique set of genes
What do transcription factors do?
proteins that bind DNA and regulate gene expression by promoting or suppressing transcription
How are transcription factors named?
- by what they do
RNA Pol vs DNA Pol
- One produces RNA, one produces DNA
- RNA poly does not require primer
- DNA faster than RNA pol
*
Protein dependent termination
- C/A rich sequence called rut site
- process continues until termination site reached
- rho protein is a helicase, binds to rut site
Protein independent termination
- 3 U’s near 3’ end of transcript
- self-complementary regions in transcript form hairpin 15-20 before 3’ end
- makes RNA poly pause, then dissociate
- C/G rich
RNA Processing steps
- Splicing out introns, leaving exons (pro/euk; mRNA)
- Addition of 5’ cap (euk mRNA)
- Addition of 3’ polyA tail (euk mRNA)
- Clevage
- Base modification
- RNA editing
rRNA processing
- in pro/euk
- created from longer pre-rRNA by cleavage/methylation
tRNA processing
- formed from pre-tRNA by cleavage, base modification, splicing
Consensus sequence for polyadenylation
- @ -10 and -30
- betweem -40 and -60
snRNA
- snRNP (small nuclear ribonuclear proteins) RNAs
- Part of spliceosome
Spliceosome
- U1 and U2 snRNPs bind intron’s ends (at 5’ and 3’ ends)
- U2-6 bind along with other proteins to splice intron
Tetrahymena
self splicing group I intron
Two ways a cell can control which genes are expressed
- Increase the affinity of the promotor for RNA polymerase
- Make promotor physically more accessible → controlling DNA structure
How do activators/repressors regulate transcription
- bind promoter/release from promoter to allow or prevent transcription of genes
Operon
- functioning unit of DNA containing cluster of genes under the control of one promoter
- Found in prokaryotes
Chromatin
- Tightly wound complex of DNA and histones to allow for control of DNA/gene expression
- Remodeling only happens in eukaryotes
What are histones?
- proteins found in nuclei that package and order the DNA into structural units called nucleosomes
Histone structure
- core: 2(H2A, H2B, H3, H4)
- H1: links nucleosomes to create chromatin
Histone modifications
- methylation
- acetylation
- Affect charge (DNA binding) or sturcture/condensation to activate or repress transcription
Enhancer
- region of DNA bound by proteins that activate transcription of a gene
Silencer
DNA sequence capable of binding repressors
Location of silencer/repressor
- Can be anywhere, but usually upstream of target gene
Location of activator/enhancer
- could be up or downstream of gene
- do not act on promoter region
tRNA
What do aminoacyl-tRNA synthetases do?
- enzyme that attaches the appropriate amino acid onto its tRNA
- By esterification of a specific cognate amino acid
How do aminoacyl-tRNA synthetases determine genetic code?
- Adds charged a.a. onto growing polypeptie chain
- tRNA reads genetic code and adds new a.a according to genetic code
Second genetic code
- matching each a.a with correct tRNA (must be specific for each other) can be viewed as second genetic code
- the “code” is in the molecular recognition of a specific tRNA molecule by a specific synthetase
Wobble
- pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules
- allows some base pairs to bind more than one codon (3rd bp of codon)
Ribosome structure
small and large subunits
Ribosome small subunit
- decoding center which monitors the complementarity of mRNA and tRNA
- has APE sites for adding polypeptides
Ribosome large subunit
- contains active site (creates peptide bonds when proteins are synthesized)
The directions in which mRNA is translated and proteins are synthesized
- 5’ to 3’
- N to C terminus
3 steps of translation
- Initiation
- Elongation
- Termination
Initiation (translation)
- mRNA and aminoacylated tRNA bind to ribosome
- IF-1, IF-2, IF-3 (initiation factors)
- FMet (forylmethothionine)
- GTP
Elongation (translation)
- Cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached
- EF-TU (elongation factor Tu)
- GTP
Termination (translation)
- mRNA and protein dissociate, ribosome recycled
- RF-1, RF-2, RF-3 (release factors)
Prokaryotes (translation)
- Shine-Dalgarno sequence
- Must be located for initiation
Eukaryotes (translation)
- Scanning
- mRNA loops through ribosome
- Binds 5’ cap
MET vs FMET
- FMET binds P site along with initiating AUG
- MET in eukaryotes
- FMET in prokaryotes
EF-Tu
- Aminoacyl tRNA binds to a complex of elongation factor Tu that also carries GTP
EF-G
- translocase
- prokaryotic elongation factor with GTPase
- catalyze the coordinated movement of tRNA and mRNA through the ribosome
- Moves tRNA along to next site
3 stop codons
- UAA
- UGA
- UAG
- Recognized by termination (release) factors/ribosome
