Genes Flashcards
What is the most common way to determine a protein sequence?
By sequencing the gene (DNA) that encodes it
What are sugars joined by?
5’ to 3’ phosphodiester linkage
Which is the beginning and which is the end of the chain in terms of 5’ and 3’
5’ start
3’ end
What bond is between complimentary bases?
Hydrogen
A + T = 2 hydrogen bonds
G + C = 3 hydrogen bonds
What does the stability of a double helix result from?
- Hydrogen bonds between pairs
- Hydrophobic effect of bases expelling water from the centre
- Van der waals forces between stacked bases
- Hydrophilic interactions of polar phosphate and water on the outside
Features of B form DNA?
- Backbone outside
- Bases inside
- Adjacent bases separated by 0.34nm
Which is more stable, DNA or RNA? Why?
DNA - hydrogen more stable than an OH group
Features of RNA polymerase
- Three major types in E. Coli
- DNA polymerase III is the major replication enzyme
- Requires a template strand
- Requires deoxynucleoside triphosphates (dNTPs = dATP, dTTP, dCTP, dGTP)
How is the lower strand replicated in DNA?
Semi discontinuous replication - lagging strand
What does DNA ligase do with the lagging strand?
Seals it
What is the difference between DNA polymerase and RNA polymerase?
RNA polymerase can initiate strand synthesis
How does RNA polymerase make a new strand?
- RNA primer made first
- DNA polymerase extends RNA strand
- Dedicated RNA polymerase: primase
(And then get rid of RNA)
What does exonuclease do?
‘Digest’ DNA - hydrolyse phosphodiester bonds
What does helicase do?
Unwinds two strands
What does single stranded binding proteins do?
Protect single stranded regions
What does topoisomerase do?
Relieves the over winding
What does primase do?
Adds an RNA primer to provide a starting point
What does RNA polymerase do?
Extends the new strand by adding nucleotides in the 5’ —> 3’ direction
What is the difference between the leading strand and lagging strand in replication?
Leading strand - synthesised continuously
Lagging strand - synthesised in short Okazaki fragments, requiring multiple primers
What does RNA polymerase I do in replication?
Replaces RNA primers with DNA
What does DNA ligase do in replication?
Joins Okazaki fragments to create a continuous strand
What are the steps of semi-conservative replication?
1- Initiation
-helicase unwinds, makes replication fork
-SSB’s stabilise strands
-Topoisomerase relieves supercooling stress
2- Elongation
-primase adds rna primer
-DNA polymerase extends strand
3- Termination
-DNA polymerase I replaces RNA primers with DNA
-DNA ligase joints Okazaki fragments
What is semi-discontinuous replication?
DNA polymerase can only synthesise DNA in 5’—>3’ direction so the lagging strand must by synthesised in fragments (Okazaki).
What is the mechanism of semi-discontinuous replication?
On lagging strand:
- runs in 3’-5’ so cant be continous
-primase lays down multiple RNA primers
-DNA polymerase synthesises short Okazaki fragments in 5’-3’ direction
-DNA ligase joins the Okazaki fragments to create continuous strand ]s
What do exonuclease’s do?
Example?
Remove nucleases one at a time from the ends of a DNA or RNA strand
Eg: DNA polymerase I - removes RNA primers in replication
Key roles of exonuclease’s?
Proofreading DNA replication
Degrading damaged or unwanted Nucleic strands
What do endonuclease’s do?
Example?
Cut within DNA/RNA strand, breaking the phosphodiester bonds inside the sequence
Eg: Restriction enzymes used in genetic engineering
Key role of endonuclease’s?
DNA repair - cutting out damaged regions
Genetic recombination
Biotechnology - gene cloning
What is Meselson and Stahl’s experiment?
- Fed E. Coli cells with heavy isotope nitrogen (^15NH4Cl)
- E. Coli made ‘heavy’ DNA
- Switched them to light (^14NH4Cl) for one generation
- Extracted the E. Coli DNA and separated on a CsCl density gradient
Steps for density gradient equilibrium sedimentation
- Put solution of Caesium chloride + DNA in a tube, and centrifuge for a long time
- Creates a density gradient in which DNA separates out
- N14 DNA will remain on the top of the vial while the ‘heavy’ N15 will sediment towards the bottom of the vial
What are ways that DNA is damaged?
- UV light
- Carcinogenic chemicals
- Free radicals
- Intrinsic chemical nature of DNA
What is Werner’s syndrome?
Defect in one of many DNA repair enzymes
RecQ helicase
-leads to premature aging and cancers
What is spontaneous deamination?
Natural process in which amine (-NH2) group is removed from a DNA base, leading to mutations if not repaired.
Changes base pairing characteristics
This process occurs without external factors and is a common form of DNA damage
What are the effects of spontaneous deamination?
- Mutations if bases are not repaired
- cpG mutation hotspots: C —> T transitions
- Cancer risk
What are some repair mechanisms for deaminated bases?
- DNA glycosylase removes the incorrect base
- AP endonuclease cuts the DNA backbone
- DNA polymerase fills the gap
- DNA ligase seals the strand
Why is the intermediate RNA step needed?
1- Amplifies the information held in DNA
2- compartmentation: info storage in nucleus, protein synthesis in cytoplasm
3- It is easier to use and control the working copy (RNA) without compromising the master copy (DNA)
4- Evolutionary remnant - RNA was the original genetic material
What is the difference between RNA polymerase and DNA polymerase?
- Does not require a primer
- No proof reading activity rate
- RNA strand does not remain hydrogen bonded to the DNA template
- Not all DNA is transcribed - only protein coding sections
What is the definition of transcription?
Transcription is the process by which RNA is synthesized from a DNA template. It occurs in three main stages: Initiation, Elongation, and Termination.
What carries out transcription?
This process is carried out by RNA polymerase and produces mRNA, rRNA, or tRNA, depending on the gene being transcribed.
What happens in the first step of transcription? (Initiation)
- RNA polymerase binds to the promoter region of the gene.
- In prokaryotes, the σ (sigma) factor helps RNA polymerase recognize the promoter.
- In eukaryotes, transcription factors (TFs) and the TATA box assist RNA polymerase II.
- DNA unwinds, forming a transcription bubble.
What happens in the second step of transcription? (Elongation)
- RNA polymerase moves along the DNA, reading the template strand (3’ → 5’) and synthesizing RNA (5’ → 3’).
- RNA polymerase adds ribonucleotides (NTPs) and catalyzes phosphodiester bond formation.
- The newly synthesized pre-mRNA (in eukaryotes) or mRNA (in prokaryotes) extends.
What happens during the third step of transcription in eukaryotes? (Termination)
✅ RNA polymerase continues past the gene and is cleaved at a polyadenylation signal (AAUAAA).
✅ The pre-mRNA undergoes processing (splicing, capping, polyadenylation) before translation.
Which is faster, transcription or DNA replication?
DNA replication
What does promoter regions contain?
Start signal sequences
What are some reasons that genes are transcribed individually?
1- precise cell regulation
2- cellular specialisation
3- alternative splicing
4- mRNA processing and stability
5- preventing unwanted mutations
Why precise gene regulation a reason why genes are transcribed individually?
• Cells need to control when and how much of a specific protein is made.
• Individually transcribed genes allow cells to turn genes on or off based on their needs.
Example: Insulin is only transcribed in pancreatic cells, while hemoglobin is transcribed in red blood cell precursors.
Why is cellular specialisation a reason why genes are transcribed individually?
-In multicellular organisms, different cells express different genes.
-Individual transcription ensures that a liver cell expresses liver-specific genes, while a neuron expresses neuron-specific genes
Why is alternative splicing a reason why genes are transcribed individually?
- Individual transcription allows for alternative splicing, where a single gene can produce multiple protein variants by splicing different exons.
- This increases protein diversity without needing more genes.
Why is mRNA processing and stability a reason why genes are transcribed individually?
- Capping, splicing, and polyadenylation require individual transcription.
- Separating gene transcription prevents interference between mRNA molecules and ensures correct modification.
Why is preventing unwanted mutations a reason why genes are transcribed individually?
- If multiple genes were transcribed together, a mutation in one gene could affect the entire set.
- Individual transcription isolates errors, making mutations less harmful overall.
What is the definition of splicing?
the process where introns (non-coding sequences) are removed from a pre-mRNA transcript, and exons (coding sequences) are joined together to form a mature mRNA molecule
When is splicing essential?
In eukaryotic gene expression before translation.
What is the third step of splicing?
- Exon joining and intron removal
- The free 3’ OH of the first exon attacks the 3’ splice site, linking the exons together.
- The lariat intron is released and later degraded.
- The mature mRNA is now ready for translation.
What is the first step of splicing?
- Recognition and assembly• The spliceosome, a complex of small nuclear RNAs (snRNAs) and proteins (snRNPs, “snurps”), recognizes specific splice sites.
• Key sequences:
○ 5’ Splice Site: GU (donor site)
○ Branch Point: Adenine (A) near the middle of the intron
○ 3’ Splice Site: AG (acceptor site)
What is the second step of splicing?
- Intron loop formation (Lariat structure)
- The branch point adenine (A) attacks the 5’ splice site, forming a loop (lariat structure).
- The 5’ end of the intron is now linked to the branch point via a 2′-5′ phosphodiester bond.
How many types of splicing is there?
What are they?
2:
Constitutive splicing
Alternative splicing
What is constitutive splicing?
Introns are always removed, and exons are joined in a fixed order.
What is alternative splicing?
Different combinations of exons are included or skipped, creating multiple mRNA variants from a single gene.
There are 4 types of alternative splicing?
What are the 4 types of alternative splicing?
Exon skipping
Intron retention
Mutually exclusive exons
Alternative 5’ or 3’ splice sites
Why is splicing important?
Enhances genetic diversity
Regulates gene expression
Prevents errors
Key reasons for RNA degradation?
- Regulation of gene expression
- Quality control and error removal
- Cellular homeostasis
- Response to environmental stress
What are the start codons for all proteins?
AUG or ATG
(Met)
What is usually the first start codon for eukaryotic translation initiation?
First AUG downstream of the mRNA cap
What is usually the first start codon in prokaryote translation initiation?
The first AUG downstream of the shine dalgarno sequence (AGGAGG)
What are the dedicated stop codons?
UAA
UGA
UAG
What is in the middle of mRNA? (structure)
Open reading frame
What are point mutations?
Substitutions - there’s a wrong base
What are indels? What is the consequence of these?
Insertions and desertions of one or more nucleotides
Consequence - frame shift mutation
What are the three types of mutations that could come from base substitutions?
Missense mutation - different amino acid
Silent mutation - same amino acid
Nonsense mutation - stop codon
Simple summery of ribosomes role in translation?
Ribosome moves along the transcript, brings in the correct amino acids, and catalyses bond formation
What are the 5 main parts of tRNA?
3 end - amino acids attaches
5 end
D loop
T loop
Anticodon loop - complimentary to mRNA codon
What attaches the amino acid to tRNA?
Specialised enzyme
What do ribosomes pair with codons?
Aminoacyl-tRNAs
What sites does a ribosome have?
3 tRNA docking sites and 1 mRNA binding site
What are the steps of the elongation cycle?
1- codon recognition: tRNA binds to A site
2- peptide bond formation: polypeptide chain grows as peptide bond is formed between the new amino acid and growing chain
3- translocation: ribosome moves forward - shifts the tRNA from the A site to P site
What ate the steps of termination of translation?
1- stop codon recognition
2- A release protein binds to stop codon in the A site
3- bond between tRNA and polypeptide chain is broken, releasing protein
4- ribosome, mRNA and tRNA separate
What are the basic steps of isolating DNA?
1 - lyse the cells (break down cell useing detergent)
2- remove other cell components (eg by using ion exchange column)
3- precipitate the DNA from solution
What are the steps to precipitate Nucleic acids?
1- break open cells using detergents or enzymes
2 - add salt (sodium acetate / NaCl) to neutralise charge - clump together
3- add cold ethanol - reduces solubility
4- let sit at cold temp and centrifuge
5- wash pellet with 70% ethanol to remove impurities. Dissolve for use
What can double helix stability be disrupted by?
- heating the DNA solution
- raising the pH
- adding chaotropic agents such as formahide (very strong H bonds)
Why does double stranded DNA absorb less UV light than single stranded DNA?
Base stacking in double helix constrains the ability of base electrons to absorb photons
What can the absorption of UV light of DNA be a good measure for?
Degree of DNA denaturation
What does each point of a DNA melting curve mean/
First straight horizontal line - strands still together
Rapid increase - in the middle = temp which DNA is 50% denatured
Second straight and horizontal line - strands are separated
What does the Tm depend on?
Base proportions in DNA
The more G:C = more energy required to denature = higher Tm
How does gel electrophoresis separate DNA?
By the size (length) - sieves the DNA and smaller DNA fragments travel faster
How do fluorescent dyes work?
Intercalates between the stacked bases
Interaction enhances fluorescence
How do you make a hot copy of DNA?
1- denature DNA to separate strands
2- add radioactive or fluorescently labeled nucleotides
3- add DNA polymerase to add these nucleotides
4- stop reactions using heat or chemical inhibitors
5- remove unused nucleotides by filtration or column purification
6- check the labeled DNA using autoradiography
What are the steps of audio radiography?
1- place sample on gel
2- expose to film - X-ray or phosphor screen over in a darkroom
3- let sit for hours to days
4- develop film using chemical developer to reveal dark spots
5- analyse
Limitations of gel electrophoresis
Upper size limit of 20,000 base pairs
Can’t separate whole chromsomes
What are restriction endonuclease
Enzymes from bacteria that cut DNA at specific recognition sites
What are the benefits of endonuclease’s?
Reduces length - more manageable
How do we detect a specific fragment (gene) in a mixture?
1 - use a prone complimentary to the fragment (southern blotting)
2 - by selectively amplifying the desired fragment (PCR)
What are the steps of exponential amplification? (PCR)
1- denature the DNA by heating (94) to separates strands
2- annealing - cool to 50-65 so primers bind to target DNA
3- extension - DNA polymerase extends new strands using dNTPs (72)
4 - repeat 25 to 40 times
What is multiplex PCR?
Variation of PCR that allows you to copy multiple DNA sequences at the same time in a single reaction
How does multiplex PCR work?
It used multiple sets of primers. Each primer set is designed to target a different DNA sequence
What is northern blotting?
Lab technique used to detect specific RNA sequences within a complex mixture of RNA
What are the steps of northern blotting?
1- RNA samples separated using gel electrophoresis
2- RNA transferred to a membrane
3 - complimentary RNA / DNA sequence is added (probe). This binds to RNA you are looking for
4- location of probe is detected
What are the steps to make cDNA?
1- isolate mRNA from cells
2- add reverse transcriptase, a primer and nucleotides. Enzyme converts mRNA to cDNA
3- treat with RNase or alkaline to degrade original RNA strand
