Nage Flashcards
How are the prime is used in a numbering of nucleic acid‘s?
1’ = attached to base
5’ attached to phosphate
Draw the structure of ribose and deoxyribose noting the differences
Pentode sugar in both w O at top 1 is H 2 is H in deoxyribose 2 is OH in ribose 3 is OH 4 is H and C(OH,H,H)
What is the structural differences between thymine and uracil?
Both have carbonyl groups
Only thymine contains methyl group (absent in uracil, only H pres)
How can you identify a cytosine from other bases
Cytosine Is the only base to contain an amino group
How can you identify a pyrimidine base
This base does not contain any amino groups or carbonyl groups or methyl groups
Amino = cytosine Carbonyl = thymine and uracil Methyl = thymine
What is a nucleoside
Only base and sugar
What is the name of the nucleoside for each of the following bases: A G C U T
(Deoxy)adenosine (Deoxy)guanosine (Deoxy)cytidine (Deoxy)uridine (Deoxy)thymidine
What is the 3/5’ end?
Phosphate group (head) =5 Ribose =3
What links the chain of nucleotides
Phosphodiester links
What do you phosphodiester links link?
OH group of ribose
O- of phosphate
How did Watson and Crick discover double helix structure
Analysis of x-ray diffraction patterns
Which way do the negative charges of DNA orient
They point outwards therefore DNA is negative
How many hydrogen bonds between the bases
Three between G and C (more stable)
2 between A and T
How do you melt and re-anneal DNA
Melt
High temperature or low salt environment
Re-anneal
Low temperature or high salt environment
What is the other word for melting and re-annealing DNA
Denaturing
Hybridising
What is different between the Genome of E.coli and human
E. coli
4.6 x 10^6 bp
Single circular double stranded molecule
Human
3 x 10^9 bp
Divided to chromosomes each containing linear DNA
How many chromosomes in diploid human cells
22 normal pairs and one pair of sex chromosomes
What is special about abnormal karyotypes
Can be extra or reduced number of individual or pairs of chromosomes e.g. trisomy 21 or translocations
How many base pairs of DNA is contained in a nucleosome
~200bp DNA (LINKER PLUS CORE DNA)
How do nucleosomes shorten DNA
7 fold condensation of DNA
DNA chain packing leads to what after 7-fold condensation of DNA?
Further 40 fold condensation
What are the levels of DNA organisation
Short region of DNA double helix (2 nm) Beads on a string form of chromatin 30 nm chromatin fibre of packed nucleosomes Chromatin fibre folded into loops Entire chromosome
What is DNA Primase
Enzyme involved in DNA replication
Catalyses the synthesis of a short RNA segment called a primer complimentary to a ssDNA (single-stranded DNA)
= type of RNA polymerase
How is the DNA double helix opened?
DNA Helicase uses energy from ATP
💔 hydrogen bonds and unwind DNA helix
What do DNA polymerases do?
Add nucleotides to 3’ end of growing chain
What things are required for Protein synthesis
Template strand
Oligonucleotide primer
Supply of dNTPs
What is the template strand also known as
Antisense
What is the sense strand
New strand being synthesised
What is an oligonucleotide primer
Short chain of nucleotides already joined, onto which new nucleotides can be added
What are deoxynucleotide triphosphates (dNTPs)?
Nucleoside triphosphates containing deoxyribose
= building blocks of DNA
Lose 2x phosphate groups when incorporated into DNA during replication
New nuclear tides are added to which end of the chain
3’ end
On three hydroxyl groups causing synthesis in the 5 to 3 direction
During the enzyme reaction when nucleotides are joined together to form the chain how is energy released ?
Energy released by triphosphate hydrolysis
What are chain terminators ?
Nucleoside analogs
Replace nucleosides yet have no hydroxyl group on the 3’, preventing addition of further bases
List examples of chain terminators
The following are nucleoside analogs:
Dideoxycytidine (HIV)
Azidothymidine (HIV)
Acyclovir (Herpes)
Where does your replication start
Origins of replication
This site is called a replication fork
Describe the asymmetry in DNA replication
Due to antiparallel strand orientation
Leading strand synthesised continuously 5 to 3
Lagging strand synthesised in short Okazaki fragments also 5 to3
How is DNA primase involved in the start of replication
DNA primase synthesises a short RNA fragment which is transient and allows replication to start at a replication origin
DNA polymerase can then add bases
How are Okazaki fragments formed
List steps
New RNA primer is required per fragment made by Primase
DNA Polymerase adds nucleotides to RNA primer to finish DNA fragment
Old RNA primer is erased and replaced by DNA
DNA ligase joins new Okazaki fragment to growing chain
What removes the old RNA primer used when forming Okazaki fragments and causes it to be erased and replaced by DNA?
Ribonuclease removes RNA primer using 5’ to 3’ exonuclease activity
Repair DNA polymerase replaces RNA with DNA
DNA ligase joins fragments using ATP to make continuous strand
What is the sliding clamp
A ring around DNA to prevent polymerase falling off
What do single strand DNA binding proteins
Prevent any localised secondary structures forming due to hydrogen bonding
What is the proofreading mechanism used during DNA replication to prevent dangerous mutations and any errors
DNA polymerase checks previous nucleotide when adding a new nucleotide for correct base pairing
If incorrect DNA polymerase’s exonuclease activity will remove it, new correct replacement is added
How is replication in E. coli different to humans
E. coli have bidirectional replication from OriC
Creates two replication folks that move simultaneously in opposite directions
Chromosomes have many replication origins each evenly spaced, giving bidirectional replication along the Strand until forks meet
Describe the M phase
Mitosis
One hour of cell division
Chromatids separate to daughter cells
Describe the G1 phase
Gap phase 1
10 hours without synthesis of DNA
RNA and proteins synthesised
DNA present as single linear double helix
Describe the S-phase
Synthesis
Nine hours and DNA synthesised
Describe the G2 phase
Gap phase 2
Four hours and between synthesis and mitosis
Makes proteins and grows
Each chromatid has two identical sister chromatids
At which stage of the cell cycle does the nucleus disintegrate
At which stage of the cell cycle does the nucleus reform
Metaphase
Reforms at interphase
How many base pairs in the human genome
6x10^9
What are housekeeping genes
Genes that are present in all cells, they are important for normal cell function & viability
What is the initial product of gene expression
ssRNA
List the three steps involved in DNA transcription
- DNA strands unwind
- Ribonucleotides base pair w bases on antisense (template) strand
- Bases joined by phosphodiester bonds, with the RNA chain growing one base @ time in the 5 to 3 direction
What is the template or antisense strand
The Strand upon which the RNA sequence is created
What are transcription factors
Special gene regulatory proteins
List the types of RNA involved in each of the following polymerase roles:
RNA Polymerase I
RNA Polymerase II
RNA Polymerase III
rRNA
Used to produce the large ribosomal subunit
mRNA
Used to produce the protein-encoding RNAs & the microRNA
tRNA / 5S RNA
Used to produce each of the tRNAs & the small ribosomal RNA subunit
What does the DNA sequence TATA specify
The initiation point for transcription after which RNA polymerase II will begin transcription
The initiation step is required to build a transcription complex before RNA synthesis occurs, where does the complex assemble
Complex assembles at gene promoter, level of transcription is regulated by activity of transcription factors
What are the steps involved in formation of the basal transcription complex
- TF IID (TATA binding protein) binds to minor groove,
Beginning to unwind DNA to allow more contact with DNA bases; asymmetric unwinding ensures unidirectional transcription - TF IIA & TF IIB bind next
- RNA Pol II binds to TF IIB w the TF IIF attached
- TF II E,H,J attach to RNA Pol II
H promotes further unwinding
How can transcription factors modulate transcription
TFs bend DNA on binding, interacting with each other and the basal transcription complex to modulate transcription
How can transcription factors remodel chromatin
Recruit proteins with enzymatic activities to modify histones
What determines transcription factor expression
Cell lineage
Altered by external signals e.g. hormones, growth factors, stress
What do mutated or abnormal transcription factors lead to
Implications in cancer and disease
How many RNA Polymerases does E. Coli have
Only have one type of RNA polymerase with all subunits
2 a , 2 B
Promoter = TATAAA
What is involved in splicing
Introns are removed
Modified nucleotide cap at 5’ end added and Poly-A tail at 3’ end
How can you distinguish introns from exons
Introns begin w GU & end w AG
Exons also end AG
Exon|Intron = AGGU
Where is the splice donor site
End 2 bases of the exon and GU of intron
What is the splice acceptor site
15 pyrimidine bases
Any base
Then CAG
(Pyr15NCAG)
What are snRNPs
Small ribonuclear proteins
Prod. from snRNA
Numbered and form spliceosome
Eg. U1, U5, U2, U4, U6
Explain the steps involved in the formation of the spliceosome
- U1 binds to splice donor site
- U5 binds to splice acceptor site
- U2,4,6 bind to middle of intron
Splicing complex forms from U1,2,4,5,6 to cleave splice donor sequence
What steps will happen after the formation of the spliceosome?
- Interval btwn Exon|Intron (AGGU) is cleaved
- An “A” residue in intron used as branchpoint to which UG bonds
- Branch results in phosphodiester bond btwn 5’ phosphate on G and 2’ OH of A (UGA)
- Phosphodiester bond btwn G (on Pyr15NCAG) and Exon cleaved
😁Lariat structure formed from Intron !
Exons=joined by RNA Ligase forming continuous sequence
What post transcriptional mRNA modification occurs?
CAP struct added 5’ end of mRNA
Poly-A tail to 3’
How does 5’ capping occur?
Post transcription:
5’ end of mRNA has a nucleotide w a triphosphate on its 5’ CArbon
The terminal phosphoanhydride bond is cleaved to form a diphosphate
The final two phosphates of GTP are removed to form GMP which then attaches to the diphosphate to form a 5-5 phosphate linkage
Finally, the 7 carbon in guanine is methylated
= 7-methylguanylate cap
What are the functions of a 5’ cap?
- prevents exonuclease degradation
- allows recognition by ribosomes
How do viruses alter euk cells to allow for only the viral mRNA to be translated in host cells
Viruses lead to enzymes being produced that remove caps
