What is the purpose of DNA repair?

To detect and correct DNA damage before mutations become permanent

What are the five major DNA repair pathways?

Mismatch repair, base excision repair (BER), nucleotide excision repair (NER), photoreactivation and translesion synthesis

What damage does BER commonly repair?

Deamination, oxidation, alkylation and depurination.

A site where the base has been removed but the sugar-phosphate backbone remains intact.

Why is uracil abnormal in DNA?

DNA normally contains thymine instead of uracil

Why does DNA use thymine instead of uracil?

So uracil produced by cytosine deamination can be recognised and removed.

What happens if an AP site is not repaired?

Translesion polymerases may insert random bases, causing mutations.

What are translesion polymerases?

Error-prone polymerases that bypass damaged DNA so replication can continue.

Which repair pathway is fast but error-prone?

Non-homologous end joining (NHEJ).

What template is used during homologous recombination?

An intact homologous DNA molecule.

What is a Holliday junction?

A crossed DNA intermediate formed during homologous recombination.

The visible site where homologous chromosomes exchange DNA during meiosis.

Why are Okazaki fragments necessary?

DNA polymerase can only synthesise DNA in the 5'→3' direction.

What is the function of single-strand binding proteins?

Prevent separated DNA strands from re-annealing.

Why are RNA primers required?

DNA polymerase cannot start DNA synthesis de novo.

Why are origins of replication AT-rich?

A-T base pairs have two hydrogen bonds, making strand separation easier.

Why do eukaryotes require multiple origins?

Their chromosomes are much larger.

What happens during proofreading?

Incorrect nucleotides are removed and replaced before synthesis continues.

Why is DNA replication highly accurate?

Correct base pairing, proofreading, and post-replication repair all reduce errors.

BER removes damaged bases first. NER removes an entire stretch of nucleotides.

HR uses a template and is accurate. NHEJ directly joins DNA ends and is error-prone.

Leading vs lagging strand

Leading = continuous. Lagging = discontinuous (Okazaki fragments).

Helicase vs topoisomerase

Helicase separates strands. Topoisomerase relieves torsional stress.

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BIOL2161 Sup

Question	Answer
What is the purpose of DNA repair?	To detect and correct DNA damage before mutations become permanent
What are the five major DNA repair pathways?	Mismatch repair, base excision repair (BER), nucleotide excision repair (NER), photoreactivation and translesion synthesis
Which repair pathway repairs replication errors?	DNA mismatch repair
Which repair pathway removes a damaged base first?	Base excision repair
Which repair pathway removes an entire nucleotide region?	Nucleotide excision repair
What damage does BER commonly repair?	Deamination, oxidation, alkylation and depurination.
What enzyme begins base excision repair?	DNA glycosylase
What is an AP site?	A site where the base has been removed but the sugar-phosphate backbone remains intact.
Which DNA polymerase mainly fills gaps during BER?	DNA polymerase β.
Why is DNA polymerase β more error-prone?	It lacks proofreading activity
Which spontaneous reaction converts cytosine into uracil?	Deamination
Why is uracil abnormal in DNA?	DNA normally contains thymine instead of uracil
Why does DNA use thymine instead of uracil?	So uracil produced by cytosine deamination can be recognised and removed.
What happens if an AP site is not repaired?	Translesion polymerases may insert random bases, causing mutations.
Which repair pathway removes pyrimidine dimers?	Nucleotide excision repair
What are translesion polymerases?	Error-prone polymerases that bypass damaged DNA so replication can continue.
Which repair pathway is the most accurate for double-strand breaks?	Homologous recombination
Which repair pathway is fast but error-prone?	Non-homologous end joining (NHEJ).
What template is used during homologous recombination?	An intact homologous DNA molecule.
Which protein performs homologous recombination in bacteria?	RecA
Which protein performs homologous recombination in eukaryotes?	Rad51
What is a Holliday junction?	A crossed DNA intermediate formed during homologous recombination.
During which process is homologous recombination used to generate genetic diversity?	Meiosis
What is a chiasma?	The visible site where homologous chromosomes exchange DNA during meiosis.
In which direction is DNA synthesised?	5' → 3'.
Which enzyme synthesises DNA?	DNA polymerase
Which strand is synthesised continuously?	Leading strand
Which strand contains Okazaki fragments?	Lagging strand
Why are Okazaki fragments necessary?	DNA polymerase can only synthesise DNA in the 5'→3' direction.
Which enzyme unwinds DNA?	Helicase
Which enzyme relieves supercoiling?	Topoisomerase
What is the function of single-strand binding proteins?	Prevent separated DNA strands from re-annealing.
Which enzyme synthesises RNA primers?	Primase
Why are RNA primers required?	DNA polymerase cannot start DNA synthesis de novo.
Which polymerase performs most DNA synthesis in bacteria?	DNA polymerase III
Which polymerase removes RNA primers in bacteria?	DNA polymerase I
Which enzyme seals nicks between DNA fragments?	DNA ligase
What bond does DNA ligase form?	Phosphodiester bond
Where does DNA replication begin?	At the origin of replication
Why are origins of replication AT-rich?	A-T base pairs have two hydrogen bonds, making strand separation easier.
How many origins of replication do bacteria usually have?	One
How many origins do eukaryotic chromosomes have?	Multiple
Why do eukaryotes require multiple origins?	Their chromosomes are much larger.
What enzyme replicates chromosome ends in eukaryotes?	Telomerase
Which proofreading activity increases replication accuracy?	3'→5' exonuclease activity.
What happens during proofreading?	Incorrect nucleotides are removed and replaced before synthesis continues.
Why is DNA replication highly accurate?	Correct base pairing, proofreading, and post-replication repair all reduce errors.
BER vs NER	BER removes damaged bases first. NER removes an entire stretch of nucleotides.
HR vs NHEJ	HR uses a template and is accurate. NHEJ directly joins DNA ends and is error-prone.
Leading vs lagging strand	Leading = continuous. Lagging = discontinuous (Okazaki fragments).
Helicase vs topoisomerase	Helicase separates strands. Topoisomerase relieves torsional stress.
DNA polymerase III vs DNA polymerase I	Pol III synthesises DNA. Pol I removes RNA primers and replaces them with DNA.
Why must DNA be packaged?	To protect genetic information while remaining accessible for replication and transcription.
What is the bacterial chromosome called?	The nucleoid
How many chromosomes does E. coli possess?	One circular chromosome
What proteins package bacterial DNA?	DNA-binding proteins and polyamines
What proteins package eukaryotic DNA?	Histones
What is chromatin?	DNA associated with histone proteins
What is a nucleosome?	Approximately 147 bp of DNA wrapped around a histone octamer.
Which histones form the nucleosome core?	Two copies each of H2A, H2B, H3 and H4.
What is the role of histone H1?	Stabilises higher-order chromatin; it is not part of the nucleosome core.
Why are histones positively charged?	They bind the negatively charged DNA backbone
What are the six levels of DNA packaging?	DNA helix → nucleosome → 30 nm fibre → loops → condensed fibre → chromatid.
What is euchromatin?	Loosely packed, transcriptionally active chromatin.
What is heterochromatin?	Densely packed, transcriptionally inactive chromatin.
Which proteins help organise chromosome loops?	Cohesin and condensin
What are telomeres?	Repetitive DNA sequences at chromosome ends.
What is the human telomere repeat?	TTAGGG.
Why can't the final RNA primer be replaced?	There is no upstream 3'-OH group for DNA polymerase to extend from.
What enzyme extends telomeres?	Telomerase
Why is telomerase called a reverse transcriptase?	It uses its own RNA template to synthesise DNA
Which cells normally express telomerase?	Stem cells, germ cells and many cancer cells.
Why do most somatic cells age?	Telomeres shorten with each cell division.
What is replicative senescence?	Permanent cell-cycle arrest after critically short telomeres.
Approximately what percentage of cancers reactivate telomerase?	About 90%
Why do cancer cells reactivate telomerase?	To maintain telomere length and divide indefinitely.
What is the Hayflick limit?	The typical limit of around 40–60 cell divisions before normal human cells become senescent.
Why are HeLa cells considered immortal?	They have high telomerase activity, allowing indefinite division.
Prokaryotic vs eukaryotic DNA packaging	Prokaryotes: circular DNA, nucleoid, supercoiling, DNA-binding proteins. Eukaryotes: linear chromosomes, histones, nucleosomes, chromatin, nucleus.
Euchromatin vs heterochromatin	Euchromatin is loosely packed and transcriptionally active; heterochromatin is tightly packed and largely inactive.
Histone octamer vs H1	The octamer (H2A, H2B, H3, H4) forms the nucleosome core, while H1 binds linker DNA and helps compact chromatin.
What enzyme performs transcription?	RNA polymerase
What is the function of sigma factor?	It recognises promoter sequence and helps RNA polymerase initiate transcription
What are the three stages of transcription?	Initiation, elongation and termination
What is intrinsic (rho-independent) termination?	Termination caused by formation of an RNA hairpin followed by destabilisation of the DNA–RNA hybrid
What protein is required for rho-dependent termination?	Rho protein
What is a rut site?	An unstructured RNA sequence where rho binds before terminating transcription
What are the four phases of translation?	Charging, initiation, elongation, and termination
Which RNA carries genetic information?	mRNA
Which RNA forms the catalytic core of the ribosome?	rRNA
Which RNA carries amino acids?	tRNA
Which codon usually starts translation?	AUG
Which amino acid initiates bacterial translation?	N-formylmethionine (fMet)
What sequence helps position the ribosome on bacterial mRNA?	The Shine–Dalgarno sequence
What is the function of aminoacyl-tRNA synthetase?	It attaches the correct amino acid to its corresponding tRNA using ATP
Which ribosomal site receives incoming tRNA?	A site
Which ribosomal site holds the growing peptide?	P site
Which ribosomal site releases empty tRNA?	E site
Which elongation factor delivers charged tRNA to the A site?	EF-Tu
Which elongation factor moves the ribosome to the next codon?	EF-G
What catalyses peptide bond formation?	Peptidyl transferase activity of the large subunit rRNA (a ribozyme)
What recognises stop codons?	Release factors (RF1 or RF2)
What is a silent mutation?	A base change that does not alter the amino acid
What is a missense mutation?	A mutation that changes one amino acid
What is a nonsense mutation?	A mutation that converts an amino acid codon into a stop codon
What is a frameshift mutation?	An insertion or deletion not divisible by three that alters the reading frame
Is the lac operon inducible or repressible?	Inducible
What molecule inactivates the lac repressor?	Allolactase
What does CAP bind?	cAMP
How are glucose and cAMP related?	High glucose → low cAMP; low glucose → high cAMP
When is lac operon expression highest?	When lactose is present and glucose is absent
What is catabolite repression?	High glucose lowers cAMP, reducing CAP activation and therefore decreasing lac operon transcription
Is the trp operon inducible or repressible?	Repressible
What molecule acts as the trp corepressor?	Tryptophan
What happens when tryptophan is abundant?	Tryptophan binds the repressor, allowing it to bind the operator and block transcription
What is attenuation?	Premature termination of trp operon transcription when tryptophan levels are high
Which hairpin causes attenuation?	The 3–4 terminator hairpin followed by U residues
Which hairpin allows transcription to continue?	The 2–3 anti-terminator hairpin
What is a pre-mRNA?	The initial transcript produced before RNA processing
What are UTRs?	Untranslated regions that regulate mRNA stability, localisation and translation
What is alternative splicing?	Production of multiple mRNAs from one pre-mRNA by using different exon combinations
Give an example of alternative exon usage	Fibronectin (FN1)
Give an example of alternative 3′ end processing	Immunoglobulin M (IgM)
Give an example of alternative promoter usage	Pax6
Which factor binds the TATA box?	TBP within TFIID
What is the role of Mediator?	It links activators to the Pol II transcription machinery
Which factor unwinds DNA during initiation?	TFIIH
What is the Rat1 model of termination?	Rat1 degrades the downstream RNA after cleavage and catches Pol II, causing termination
What is unusual about the 5′ cap linkage?	It is a 5′–5′ triphosphate bond
What base is added during capping?	Guanosine
What sequence signals polyadenylation?	AAUAAA
Name two functions of the poly-A tail	mRNA stability and enhanced translation initiation
What are the canonical splice-site signals?	GU … A … AG
Which snRNP binds the 5′ splice site?	U1
Which snRNP positions the branch-point adenine?	U2
Which snRNP aligns the exons for ligation?	U5
Which snRNP forms the catalytic core with U2?	U6
What structure is produced after the first transesterification reaction?	The lariat intron
Which ribosomal subunits form the eukaryotic ribosome?	40S and 60S
What sequence helps the ribosome recognise the start codon?	The Kozak sequence
What is leaky scanning?	Bypassing a weak AUG and initiating at a downstream AUG
Which proteins bind the poly-A tail?	PABPs (poly-A binding proteins)
How did Mendel define a gene?	A hereditary unit (or factor) that determines an inherited trait
What is the molecular definition of a gene?	A DNA sequence that is transcribed into an RNA molecule
What is the functional definition of a gene?	A DNA sequence together with its regulatory elements that produces one or more functional RNA molecules or proteins.
Why is the functional definition broader than the molecular definition?	Because it includes promoters, enhancers, and other regulatory sequences required for correct gene expression.
Which type of organism commonly contains introns?	Eukaryotes
Are prokaryotic genes usually interrupted by introns?	No. They usually have a continuous coding sequence
What is an operon?	A group of prokaryotic genes transcribed together as a single mRNA
Do eukaryotes commonly have operons?	No
Where does transcription occur in eukaryotes?	In the nucleus
Where does translation occur in eukaryotes?	In the cytoplasm
Why are transcription and translation coupled in bacteria?	Because bacteria lack a nucleus
Give one structural difference between prokaryotic and eukaryotic genes.	Eukaryotic genes contain exons and introns, whereas prokaryotic genes usually do not
What is an exon?	A region of a gene that remains in the mature mRNA after splicing
What is an intron?	A non-coding region removed from pre-mRNA during RNA splicing
Are all exons protein coding?	No. Some exons form part of the 5' or 3' untranslated regions (UTRs)
What is the function of the 5' UTR?	It helps regulate translation initiation and mRNA stability
What is the function of the 3' UTR?	It regulates mRNA stability, localisation and translation efficiency
Which region contains the start codon?	The coding region immediately after the 5' UTR
Which region contains the stop codon?	The end of the coding region before the 3' UTR
Which RNA polymerase synthesises mRNA in eukaryotes?	RNA polymerase II
What is the product immediately after transcription?	Pre-mRNA
What are the three major RNA processing steps?	5' capping, RNA splicing, polyadenylation
What is added during 5′ capping?	A 7-methylguanosine cap
Why is the 5′ cap important?	It protects mRNA from degradation, promotes nuclear export, and helps recruit the ribosome.
What is added during polyadenylation?	A poly(A) tail
What are two functions of the poly(A) tail?	It increases mRNA stability and enhances translation
What is pre-mRNA?	The initial RNA transcript containing both exons and introns
What is mature mRNA?	Processed RNA that has a 5′ cap, joined exons, and a poly(A) tail and is ready for translation.
Which molecule contains introns: pre-mRNA or mature mRNA?	Pre-mRNA
What is alternative splicing?	The production of different mature mRNAs by joining different combinations of exons from the same pre-mRNA.
What is the major advantage of alternative splicing?	It allows one gene to produce multiple protein isoforms.
Why is alternative splicing important in humans?	It greatly increases protein diversity without increasing the number of genes.
Can different tissues splice the same gene differently?	Yes
What is alternative exon usage?	Inclusion or exclusion of specific exons to produce different mRNA transcripts.
How does alternative exon usage affect proteins?	It changes the amino acid sequence and therefore the structure and function of the protein.
What is alternative 3′ end processing?	The use of different polyadenylation sites to generate different mRNA 3′ ends.
What can alternative polyadenylation change?	The length of the 3′ UTR and, in some cases, the protein produced.
What is alternative promoter usage?	The use of different promoters to produce transcripts with different transcription start sites.
How can alternative promoter usage affect gene expression?	Different promoters can be active in different tissues or developmental stages.
What is a protein isoform?	A different version of a protein produced by the same gene
Name three mechanisms that generate protein isoforms.	Alternative splicing, alternative promoter usage, alternative 3' end processing
Which RNA-processing mechanism is illustrated by fibronectin?	Alternative splicing
Why does fibronectin undergo alternative splicing?	Different tissues require different fibronectin protein isoforms.
What are the two major forms of fibronectin?	Plasma fibronectin and cellular fibronectin
Which RNA-processing mechanism is illustrated by IgM?	Alternative 3' end processing (alternative polyadenylation)
What are the two forms of IgM?	Membrane-bound IgM and secreted IgM
How are membrane-bound and secreted IgM produced from the same gene?	By using different polyadenylation sites during RNA processing.
Which RNA-processing mechanism is illustrated by Pax6?	Alternative promoter usage
Why does Pax6 use alternative promoters?	To allow tissue-specific and developmental regulation of gene expression.
A mutation prevents introns from being removed. What happens?	Mature mRNA cannot form correctly, so translation may produce an abnormal or non-functional protein.
Why does a single human gene often produce many proteins?	Because RNA processing can generate multiple RNA scripts from one original pre-mRNA
Which occurs first: transcription or RNA processing?	Transcription begins first, and RNA processing occurs co-transcriptionally and continues after transcription.
How many RNA polymerases do prokaryotes have?	One RNA polymerase that transcribes all RNA types
How many major RNA polymerases do eukaryotes have?	Three: RNA polymerase I, II, and III
Where does transcription occur in prokaryotes?	In the cytoplasm
Where does transcription occur in eukaryotes?	In the nucleus
Why can transcription and translation occur simultaneously in prokaryotes?	Because prokaryotes lack a nucleus
Why are transcription and translation separated in eukaryotes?	Because transcription occurs in the nucleus and translation occurs in the cytoplasm
Do prokaryotic mRNAs usually undergo extensive processing?	No
Name three major RNA processing events that occur in eukaryotes.	5' capping, RNA splicing, 3' cleaving and polyadenylation
Which RNA polymerase synthesises most rRNA?	RNA polymerase I
Which rRNAs are produced by RNA polymerase I?	28S, 18S and 5.8S rRNA.
Which RNA polymerase synthesises mRNA?	RNA polymerase II
Which other RNAs are synthesised by RNA polymerase II?	Many snRNAs and long non-coding RNAs.
Which RNA polymerase synthesises tRNA?	RNA polymerase III
Which RNA polymerase synthesises 5S rRNA?	RNA polymerase III.
Which RNA polymerase is most important for protein-coding genes?	RNA polymerase II
What is the function of a promoter?	It is the region where transcription begins
What DNA sequence is commonly found in many eukaryotic promoters?	The TATA box
Approximately where is the TATA box located relative to the transcription start site?	About 25 base pairs upstream
What does TBP stand for?	TATA-binding protein
Which transcription factor contains TBP?	TFIID
What is the first major event in transcription initiation?	TBP binds the TATA box.
Why is TBP important?	It bends the DNA and initiates assembly of the transcription machinery.
What is the pre-initiation complex (PIC)?	A complex of RNA polymerase II and general transcription factors assembled at the promoter.
Which general transcription factors help form the PIC?	TFIIA, TFIIB, TFIID, TFIIF, TFIIE and TFIIH.
What is the purpose of the PIC?	To correctly position RNA polymerase II for transcription initiation
What are activator proteins?	Regulatory proteins that increase transcription by binding enhancer sequences.
What is the Mediator complex?	A large protein complex that links activators to RNA polymerase II and the general transcription machinery.
Why is Mediator important?	It transmits regulatory signals from activators to RNA polymerase II, increasing transcription efficiency.
Can activators usually stimulate transcription effectively without Mediator?	No, Mediator is required to communicate many activation signals.
Which transcription factor unwinds DNA during initiation?	TFIIH
What enzymatic activity allows TFIIH to open DNA?	Helicase activity
What is the second important function of TFIIH?	It phosphorylates the C-terminal domain (CTD) of RNA polymerase II.
Why is phosphorylation of the CTD important?	It allows RNA polymerase II to leave the promoter and begin elongation.
During elongation, which DNA strand does RNA polymerase II read?	The template strand (3′→5′).
In which direction is RNA synthesised?	5′→3′.
What is produced during transcription elongation?	A pre-mRNA transcript
What happens to DNA behind RNA polymerase II?	The DNA double helix reforms
Which RNA sequence signals cleavage of the transcript?	AAUAAA
What happens after the RNA is cleaved?	A poly(A) tail is added to the upstream RNA.
Does RNA polymerase II stop immediately after cleavage?	No, it continues transcribing downstream
What is Rat1?	A 5′→3′ exonuclease involved in transcription termination.
Describe the Rat1 (torpedo) model of transcription termination.	Rat1 degrades the downstream RNA after cleavage, catches RNA polymerase II, and promotes its release from the DNA.
When is the 5′ cap added?	Shortly after transcription begins (co-transcriptionally).
What nucleotide forms the 5′ cap?	7-methylguanosine
What is unusual about the 5′ cap linkage?	It is attached by a 5'-5' triphosphate bond
Give three functions of the 5′ cap.	Protects mRNA from degradation, promotes nuclear export, facilitates translation initiation
Which translation initiation factor recognises the 5′ cap?	eIF4E.
What is RNA splicing?	The removal of introns and joining of exons
Where does RNA splicing occur?	In the nucleus
Which complex carries out RNA splicing?	The spliceosome
Why is splicing necessary?	To produce a continuous coding sequence for translation
What sequence signals 3′ cleavage?	AAUAAA
What enzyme adds the poly(A) tail?	Poly(A) polymerase
Approximately how many adenine nucleotides are added?	About 200
What is the poly(A) tail?	A stretch of adenine nucleotides added to the 3′ end of mRNA.
Give three functions of the poly(A) tail.	Protects mRNA from degradation, increases mRNA stability, enhances translation efficiency
Which proteins bind the poly(A) tail?	Poly(A)-binding proteins (PABPs)
How do PABPs promote translation?	They interact with cap-binding proteins to form a closed-loop mRNA, improving ribosome recruitment.
What is pre-mRNA?	The initial transcript containing exons and introns
What is mature mRNA?	The fully processed transcript with a 5' cap, joined exons, and a poly(A) tail
Which form contains introns?	Pre-mRNA
Which form is exported from the nucleus?	Mature mRNA
Why can't pre-mRNA normally be translated?	It still contains introns and has not completed RNA processing.
What would happen if the 5′ cap were not added?	The mRNA would be less stable, exported less efficiently, and translated poorly.
What would happen if polyadenylation failed?	The mRNA would be unstable, degraded more rapidly, and translated less efficiently.
What would happen if TFIIH could no longer phosphorylate RNA polymerase II?	RNA polymerase II would fail to transition from initiation to elongation, greatly reducing transcription.
What would happen if Rat1 were non-functional?	RNA polymerase II would not terminate efficiently and would continue transcribing beyond the normal termination site.
What does "co-transcriptional processing" mean?	RNA processing that occurs while RNA polymerase II is still transcribing RNA
What are the three major co-transcriptional processing events?	5' capping, RNA splicing, and 3' cleavage and polyadenylation
Which RNA polymerase produces transcripts that undergo these processing steps?	RNA polymerase II
Why is RNA processing necessary?	To produce a stable, mature mRNA that can be exported from the nucleus and translated into protein.
When is the 5′ cap added?	Shortly after transcription begins, when the RNA is about 20–30 nucleotides long.
What molecule forms the 5′ cap?	7-methylguanosine (m⁷G).
What type of bond attaches the 5′ cap?	A 5′–5′ triphosphate linkage.
Why is the 5′–5′ linkage unusual?	Most nucleotides are linked by 3′–5′ phosphodiester bonds.
Give four functions of the 5′ cap.	Prevents mRNA from degradation, promotes nuclear transport, recruits translation initiation factors, enhances translation initiation
What is RNA splicing?	The removal of introns and the joining exons
Which complex carries out RNA splicing?	The spliceosome
What are snRNPs?	Small nuclear ribonucleoproteins that form the spliceosome.
Which snRNP recognises the 5′ splice site?	U1
Which snRNP binds the branch point?	U2
Which snRNPs join later to complete the spliceosome?	U4, U5 and U6
What is the function of U5?	It helps align the exons for ligation
What are the three essential sequence elements required for splicing?	5' splice site, branch point, 3' splice site
Which nucleotides are typically found at the 5′ splice site?	GU
Which nucleotide is found at the branch point?	Adenine (A).
Which nucleotides are typically found at the 3′ splice site?	AG
What mnemonic helps remember splice site sequences?	GU → A → AG
What is the first step of spliceosome assembly?	U1 binds the 5′ splice site.
What is the second step?	U2 binds the branch point adenine.
What happens after U4/U5/U6 join?	The spliceosome becomes catalytically active.
What happens during the first transesterification reaction?	The branch point adenine attacks the 5′ splice site, forming a lariat
What is a lariat?	A looped intron intermediate formed during splicing.
What happens during the second transesterification reaction?	The upstream exon attacks the 3′ splice site, joining the exons and releasing the intron.
What is the final product of RNA splicing?	Mature RNA with exons joined together.
What is alternative splicing?	The production of different mature mRNAs by joining different combinations of exons.
Why is alternative splicing important?	It allows one gene to perform multiple protein isoforms
What are SR proteins?	Proteins that promote exon inclusion by helping recruit the spliceosome.
What are hnRNP proteins?	Proteins that often promote exon skipping by inhibiting spliceosome assembly.
Why do different tissues produce different splice variants?	Because they express different splicing regulatory proteins.
Where does translation occur in eukaryotes?	In the cytoplasm.
Which ribosomal subunits make up the eukaryotic ribosome?	40S and 60S.
What is the complete eukaryotic ribosome called?	The 80S ribosome.
What sequence recruits ribosomes in prokaryotes?	The Shine–Dalgarno sequence.
What structure recruits ribosomes in eukaryotes?	The 5' cap
Do bacterial ribosomes scan for the start codon?	No
Do eukaryotic ribosomes scan for the start codon?	Yes
What sequence helps identify the correct start codon in eukaryotes?	The Kozak sequence
Which initiation factor binds the 5′ cap?	eIF4E.
Which proteins bind the poly(A) tail?	Poly(A)-binding proteins (PABPs).
Why do the cap and poly(A) tail interact?	To form a closed-loop mRNA that promotes efficient translation.
Which ribosomal subunit binds first?	The 40S subunit
What accompanies the 40S ribosomal subunit?	The initiator Met-tRNA and initiation factors.
What does the 40S ribosomal subunit do after binding?	Scans from the 5′ end toward the 3′ end until it finds AUG.
What is the Kozak sequence?	A consensus sequence surrounding AUG that helps identify the correct start codon.
Which codon usually initiates translation?	AUG
Which ribosomal subunit joins after AUG is recognised?	The 60S subunit.
What begins after the 80S ribosome is assembled?	Translation elongation
Name four functions of the 5′ cap.	Protects mRNA from degradation, promotes nuclear export, recruits eIF4E, initiates translation
Which initiation factor specifically recognises the 5′ cap?	eIF4E.
Which proteins bind the poly(A) tail?	Poly(A)-binding proteins (PABPs).
Give four functions of the poly(A) tail.	Protects mRNA from degradation, increases mRNA stability, promotes nuclear export, enhances translation efficiency
Why does forming a closed-loop mRNA increase translation?	It allows ribosomes to be efficiently recycled and stabilises interactions between the 5′ and 3′ ends of the mRNA.
What is the overall purpose of translation initiation?	To correctly identify the start codon (AUG) and assemble a functional 80S ribosome so protein synthesis can begin
Where does translation occur in eukaryotic cells?	In the cytoplasm
What molecule is translated into protein?	Mature mRNA
Which ribosomal subunit binds the mRNA first?	The 40S ribosomal subunit
Which amino acid is carried by the initiator tRNA?	Methionine (Met)
What is the initiator tRNA called?	Initiator Met-tRNA
Why is initiator Met-tRNA special?	It specifically recognises the start codon (AUG) during translation initiation
What is the function of the eIF4F complex?	It binds the 5' cap and recruits the translation initiation machinery
Which protein within eIF4F binds directly to the 5' cap?	eIF4E
What is the role of eIF4G?	It acts as a scaffold, linking the cap-binding complex to other initiation factors and the ribosome
What is the role of eIF4A?	It unwinds RNA secondary structure to allow ribosomal scanning
Where does the translation machinery initially bind the mRNA?	The 5' cap
Does the ribosome bind directly to the AUG start codon?	No. It binds to the 5' cap first and then scans to find the AUG start codon
What does the 40S ribosomal subunit do after binding the 5' cap?	It scans along the mRNA in the 5′→3′ direction.
What is the purpose of scanning?	To locate the correct AUG start codon
What is the Kozak sequence?	A consensus sequence surrounding the start codon that promotes efficient translation initiation
What is the Kozak consensus sequence?	GCC(A/G)CCAUGG.
Why is the Kozak sequence important?	It helps the ribosome identify the correct AUG start codon
What happens if the Kozak sequence is strong?	Translation initiates efficiently
What happens if the Kozak sequence is weak?	The ribosome may skip the AUG start codon and continue scanning (leaky scanning)
What is leaky scanning?	Failure of the ribosome to initiate at a weak AUG, causing it to continue scanning for another AUG
Why can leaky scanning be biologically useful?	It allows different proteins to be produced by the same mRNA
Which codon normally initiates translation?	AUG
What happens when the ribosome reaches a suitable AUG?	Initiator Met-tRNAᵢ base-pairs with the AUG codon.
Why is base-pairing between Met-tRNAᵢ and AUG important?	It confirms the correct translation start site
What happens to initiation factors after the start codon is recognised?	They are released from the ribosome
Why are initiation factors released?	Their role in initiation is complete, allowing elongation to proceed
Which ribosomal subunit joins after AUG recognition?	The 60S large ribosomal subunit
What ribosome is formed after the 60S subunit joins?	The 80S ribosome
Why is it called an 80S ribosome rather than 100S?	Svedberg units measure sedimentation rate, not size, so they are not additive
What happens after the 80S ribosome forms?	Translation elongation begins
During elongation, what does the ribosome synthesise?	A polypeptide chain
Name four functions of the 5′ cap.	Protects mRNA from degradation, recruits eIF4E, promotes ribosome bindings, and enhances translation initiation
Which initiation factor recognises the 5′ cap?	eIF4E
Which proteins bind the poly(A) tail?	Poly(A) bindings proteins (PABPs)
What are four functions of the poly(A) tail?	Protects mRNA from degradation, increases mRNA stability, promotes translation, and facilitates ribosome recycling
Why do the 5′ cap and poly(A) tail interact?	To form a closed-loop mRNA that improves translation efficiency
How do prokaryotic ribosomes locate the start codon?	They bind directly to the Shine-Dalgarno sequence
How do eukaryotic ribosomes locate the start codon?	They bind the 5′ cap and scan to the first suitable AUG.
Which sequence is used in bacteria instead of the Kozak sequence?	The Shine-Dalgarno sequence
What is promoter-proximal pausing?	A regulatory mechanism in which RNA polymerase II pauses approximately 20–60 nucleotides after transcription begins before entering productive elongation.
Approximately how far does RNA polymerase II transcribe before pausing?	About 20-60 nucleotides
Why do cells use promoter-proximal pausing?	To allow rapid activation of gene expression in response to signals
Which genes commonly exhibit promoter-proximal pausing?	Developmental genes and stress response genes
Which protein complex promotes promoter-proximal pausing?	NELF (negative elongation factor)
What is the role of DSIF?	It stabilises the paused RNA polymerase II complex
Which kinase releases promoter-proximal pausing?	P-TEFb
What does P-TEFb phosphorylate?	RNA polymerase II, NELF, and DSIF
What happens after P-TEFb phosphorylates these proteins?	NELF dissociates, DSIF promotes elongation, and RNA polymerase II resumes transcription
What is coordinate regulation?	The simultaneous regulation of multiple genes by the same transcription factor or regulatory pathway.
Why is coordinate regulation important?	It allows cells to produce multiple proteins needed for the same biological process at the same time.
Give an example of coordinate regulation.	Muscle-specific transcription factors activating genes encoding actin, myosin, and other muscle proteins.
What DNA elements commonly mediate coordinate regulation?	Enhancers
How can one transcription factor regulate many genes?	By binding the same enhancer sequence present near multiple genes
What is RNA interference (RNAi)?	A post-transcriptional mechanism that silences gene expression by degrading mRNA or preventing its translation.
Does RNA interference affect DNA or mRNA?	mRNA
What is the outcome of RNA interference?	Reduced or absent protein production
What does miRNA stand for?	MicroRNA
Where do miRNAs originate?	They are encoded by the cell's own genome (endogenous)
What is the primary role of miRNAs?	Regulation of normal gene expression
Do miRNAs usually bind perfectly to their target mRNA?	No, they usually bind imperfectly
What usually happens when miRNA binds its target?	Translation is repressed and/or the mRNA is destabilised and degraded.
What does siRNA stand for?	Small interfering RNA
Where do siRNAs usually originate?	Foreign double-stranded RNA (e.g. viruses) or experimentally introduced RNA.
Do siRNAs usually bind perfectly to their target mRNA?	Yes
What happens after siRNA binds its target?	Argonaute cleaves the target mRNA
Which RNA is endogenous: miRNA or siRNA?	miRNA
Which RNA commonly originates from viruses?	siRNA
Which RNA usually has imperfect complementarity?	miRNA
Which RNA usually has nearly perfect complementarity?	siRNA
Which RNA mainly represses translation?	miRNA
Which RNA mainly causes mRNA cleavage?	siRNA
What is Dicer?	An RNase enzyme that cuts long double-stranded RNA into small RNA molecules.
Approximately how long are the RNAs produced by Dicer?	About 21-23 nucleotides
Why is Dicer essential for RNA interference?	It generates the small guide RNAs required for gene silencing.
What does RISC stand for?	RNA-induced silencing complex
What is the function of RISC?	To use a guide RNA to recognise complementary target mRNA
Which protein performs the catalytic activity within RISC?	Argonaute
What is the role of Argonaute?	It binds the guide RNA and cleaves complementary target mRNA when base pairing is extensive.
What happens when complementarity is only partial?	Argonaute represses translation rather than cleaving the mRNA.
Put the RNA interference pathway in order.	Double-stranded RNA or miRNA precursor forms. Dicer cuts it into small RNAs. Small RNA loads into RISC. One strand becomes the guide strand. Argonaute recognises target mRNA. Translation is inhibited or the mRNA is cleaved.
What are post-translational modifications (PTMs)?	Chemical modifications made to proteins after translation.
Why are PTMs important?	They regulate protein activity, stability, localisation, and interactions.
What is phosphorylation?	Addition of a phosphate group to a protein
Which amino acids are commonly phosphorylated?	Serine Threonine Tyrosine
What enzyme adds phosphate groups?	Protein kinases
Name two functions of phosphorylation.	Activates or inhibits proteins and regulates cell signalling
What is acetylation?	Addition of an acetyl group, often to lysine residues
How does histone acetylation usually affect transcription?	It increases transcription by opening chromatin
What is methylation?	Addition of methyl groups to proteins or DNA
Can methylation activate or repress gene expression?	Yes, depending on the site and context
What is ubiquitination?	Attachment of ubiquitin to a protein
What is the main function of ubiquitination?	It targets proteins for degradation by the proteasome
What is glycosylation?	Addition of carbohydrate groups to proteins
Name three functions of glycosylation.	Protein folding, protein stability, and cell recognition
What is CRISPR-Cas9?	A genome-editing system adapted from the bacterial immune system.
What directs Cas9 to the target DNA?	Guide RNA (gRNA)
What is the function of Cas9?	It cuts both strands of DNA at the target site
What type of DNA break does Cas9 produce?	A double-strand break
What are the two major pathways that repair Cas9-induced DNA breaks?	Non-homologous end joining (NHEJ) Homology-directed repair (HDR)
Which repair pathway is error-prone?	NHEJ
What is a common outcome of NHEJ?	Small insertions or deletions (indels) that often knock out gene function.
Which repair pathway allows precise genome editing?	HDR
What is required for HDR?	A repair template with the desired DNA sequence
Put the CRISPR-Cas9 mechanism in order.	Design guide RNA. Guide RNA binds Cas9. Guide RNA base-pairs with target DNA. Cas9 creates a double-strand break. DNA is repaired by NHEJ or HDR.
What is a recombinant protein?	A protein produced from a gene that has been artificially inserted into another organism
What is recombinant protein expression?	The production of a protein from a recombinant DNA construct in a host organism such as E. coli.
Why is recombinant protein expression important?	It allows the large-scale production of proteins for medicine, industry and research
Give three examples of recombinant proteins used in medicine.	Human insulin, human growth hormone, and blood clotting factors
Why is recombinant insulin preferred over insulin extracted from animals?	It is identical to human insulin, can be produced in large quantities, has a lower risk of contamination, and reduces ethical concerns
Give three research applications of recombinant proteins.	Studying enzyme function Determining protein structure Investigating gene mutations
Give two industrial applications of recombinant proteins.	Food-processing enzyme and laundry detergent enzyme
Why is E. coli commonly used for recombinant protein expression?	Fast-growing, inexpensive, easy to culture, easy to genetically manipulate, and produces large amount of protein
What is one limitation of expressing eukaryotic proteins in E. coli?	E. coli cannot perform many eukaryotic post-translational modifications (e.g. glycosylation), so some proteins may not fold or function correctly.
What is the first step in recombinant protein expression?	Clone the gene of interest into an expression plasmid
What happens after the plasmid is constructed?	It is introduced into E. coli by transformation
What is transformation?	The uptake of foreign DNA (such as a plasmid) into bacterial cells
Why are bacteria grown before inducing protein expression?	To produce a large population of cells before diverting resources to recombinant protein production.
What chemical is commonly used to induce recombinant protein expression?	IPTG
What happens after IPTG is added?	Recombinant protein expression begins
What is the final step of recombinant protein production?	Purification of the recombinant protein
What is an expression plasmid?	A circular DNA molecule engineered to express a recombinant gene in a host cell
What is the purpose of the origin of replication (ori)?	It allows the plasmid to replicate independently inside host cells
Why does an expression plasmid contain an antibiotic resistance gene?	To allow selection of bacteria that contain the plasmid
What is the function of the multiple cloning site (MCS)?	It provides restriction enzyme sites where the gene of interest can be inserted.
What is the function of the promoter?	It initiates transcription of the recombinant gene
What is the function of the transcription terminator?	It stops transcription at the end of the gene
Why are purification tags (e.g. His-tags) often added?	They allow easy purification of the recombinant protein using affinity chromatography
What is LacI?	A repressor protein that blocks transcription by binding the lac operator
What happens when LacI is bound to the operator?	Transcription is repressed
What molecule is commonly used to remove LacI from the operator?	IPTG
Why is IPTG used instead of lactose?	IPTG is not metabolised by E. coli, so it provides stable and sustained induction
What happens after IPTG binds LacI?	LacI changes shape and releases the operator, allowing transcription.
What is T7 RNA polymerase?	A highly efficiency RNA polymerase derived from bacteriophage T7
Which promoter is recognised by T7 RNA polymerase?	The T7 promoter
Why is T7 RNA polymerase used instead of E. coli RNA polymerase?	It produces much higher levels of transcription
What controls expression of T7 RNA polymerase in many expression systems?	The lac operon
What happens when IPTG is absent?	LacI represses expression of T7 RNA polymerase, so the recombinant gene is not transcribed.
What happens when IPTG is added?	LacI is inactivated, T7 RNA polymerase is produced, and the recombinant gene is transcribed.
Why can't the recombinant gene be transcribed without T7 RNA polymerase?	Because the T7 promoter is recognised by the T7 RNA polymerase, not by the normal E. coli RNA polymerase
Why is recombinant protein expression usually induced rather than expressed continuously?	Continuous expression can slow bacterial growth and toxic proteins may harm or kill the cells.
Why is it beneficial to grow bacteria before inducing protein expression?	It maximises cell numbers before directing cellular resources toward producing the recombinant protein.
What is p53?	A tumour suppressor protein that acts as a sequence-specific transcription factor and is known as the 'guardian of the genome'
Why is p53 called the "guardian of the genome"?	Because it prevents cells with damaged DNA from continuing to divide by promoting cell cycle arrest, DNA repair, senescence, or apoptosis
What type of protein is p53?	A transcription factor
What does p53 bind to in DNA?	Specific DNA sequences called p53 response elements
What is the main role of p53 as a transcription factor?	To activate or repress transcription of genes involved in maintaining genome integrity.
What activates p53?	Cellular stress, particularly DNA damage
Name four types of stress that can activate p53.	DNA damage, UV radiation, oxidative stress and oncogene activation
What are the four major cellular outcomes of p53 activation?	Cell cycle arrest, DNA repair, senescence and apoptosis
Why does p53 induce cell cycle arrest?	To prevent damaged DNA from being replicated before it is repaired
Which p53 target gene is responsible for cell cycle arrest?	p21 (CDKN1A).
What does p21 inhibit?	Cyclin-CDK complexes
What is the result of cyclin-CDK inhibition?	Cell cycle progression stops, usually at the G1/S checkpoint
Why does p53 activate DNA repair genes?	To allow damaged DNA to be repaired before the cell continues dividing
What happens if DNA repair is successful?	The cell cycle can resume
What is cellular senescence?	A permanent state in which cells remain alive but no longer divide
Why is senescence beneficial?	It prevents damaged cells from becoming cancerous
What is apoptosis?	Programmed cell death
When does p53 trigger apoptosis?	When DNA damage is too severe to repair
Name three important pro-apoptotic genes activated by p53.	BAX, PUMA, and NOXA
Why does apoptosis help prevent cancer?	It removes cells with potentially dangerous mutations
Why are p53 levels normally low?	To allow healthy cells to continue dividing
Which protein negatively regulates p53?	MDM2
What type of protein is MDM2?	An E3 ubiquitin ligase
What does MDM2 do to p53?	It ubiquitinates p53, targeting it for degradation by the proteasome.
What happens to p53 under normal conditions?	It is continuously degraded by MDM2
Which kinases are activated by DNA damage?	ATM and ATR
What do ATM and ATR do to p53?	They phosphorylate p53
Why is phosphorylation of p53 important?	It prevents MDM2 from binding, allowing p53 to accumulate
What happens when p53 accumulates?	It activates transcription of target genes
Why is post-translational regulation of p53 advantageous?	It allows rapid activation of existing p53 protein without waiting for new protein synthesis.
Name three post-translational modifications that regulate p53.	Phosphorylation, acetylation, and ubiquitination
Which modification usually stabilises p53 after DNA damage?	Phosphorylation
Which modification targets p53 for degradation?	Ubiquitination
What is alternative splicing?	The production of different mature mRNAs from the same gene by including or excluding different exons.
What does alternative splicing of TP53 produce?	Multiple p53 protein isoforms
Why are different p53 isoforms important?	They can have different regulatory functions and biological activities.
What might differ between p53 isoforms?	DNA-binding ability Transcriptional activity Effects on apoptosis Effects on cell cycle regulation
Why is p53 considered a tumour suppressor?	It prevents damaged cells from dividing and surviving
What happens if p53 is mutated?	Cells with DNA damage continue dividing, allowing mutations to accumulate
Approximately what proportion of human cancers contain TP53 mutations?	About 50%
Why does loss of p53 increase genomic instability?	Damaged DNA is not repaired or eliminated before replication
What is the relationship between p53 dysfunction and cancer?	Loss of p53 removes important checkpoints that often suppress tumour formation
Why is the p53 pathway an important target for cancer therapy?	Because restoring or enhancing p53 function can suppress tumour growth.
Name four therapeutic strategies involving the p53 pathway.	Restore mutant p53 function Inhibit MDM2 TP53 gene therapy Use DNA-damaging therapies that activate p53
Why can MDM2 inhibitors increase p53 activity?	They prevent MDM2 from degrading p53
Why may tumours with mutant p53 respond poorly to chemotherapy?	They cannot effectively activate apoptosis after DNA damage.
Why are genetic modification (GM) and genome editing important in modern agriculture?	They allow crops to be improved more quickly and precisely than traditional breeding, helping address food security, climate change, pests, diseases, and environmental stress.
Name five challenges facing modern agriculture.	Climate change Drought Plant diseases Insect pests Growing global population
What are the major goals of crop biotechnology?	Increase crop yield Improve nutritional value Increase pest resistance Increase disease resistance Improve tolerance to drought, heat, or salinity
How does traditional plant breeding work?	By crossing two plants with desirable traits and selecting offspring with the best combination of traits.
What is one major limitation of traditional breeding?	Thousands of genes are inherited together, making the process slow and less precise
How is genetic modification more precise than traditional breeding?	It introduces or modifies a specific gene rather than mixing entire genomes
What is genetic modification (GM)?	The introduction of foreign DNA into an organism's genome to produce a desired trait
What is a transgene?	A gene transferred from another organism into the genome of a recipient organism.
What is transgenesis?	The process of introducing a transgene into another organism
Does transgenesis usually involve DNA from another species?	Yes
Give an example of a pest-resistant GM crop.	Bt cotton
What bacterial gene is inserted into Bt cotton?	A gene from Bacillus thuringiensis (Bt).
What advantage does Bt cotton provide?	It produces an insecticidal protein, reducing insect damage and pesticide use.
Give an example of a nutritionally enhanced GM crop.	Golden rice
Why was Golden Rice developed?	To produce β-carotene (a precursor of vitamin A) and help reduce vitamin A deficiency.
Give an example of a herbicide-tolerant GM crop.	Glyphosate-resistant soybean
Which bacterium is commonly used to make transgenic plants?	Agrobacterium tumefaciens.
Why is Agrobacterium tumefaciens useful in plant biotechnology?	It naturally transfers DNA into plant cells.
What plasmid is modified during Agrobacterium-mediated transformation?	The Ti (tumour-inducing) plasmid.
What is inserted into the Ti plasmid?	The gene of interest
What happens after the modified Ti plasmid is introduced into Agrobacterium?	The bacterium infects plant tissue and transfers the DNA into plant cells.
What happens to the transferred DNA?	It integrates into the plant genome
What is the result after DNA integration?	The plant cell becomes genetically modified
What is totipotency?	The ability of a single plant cell to regenerate into a complete plant
Why is totipotency important in plant biotechnology?	It allows an entire transgenic plant to be regenerated from one transformed cell.
Why are selectable marker genes included in plant transformation experiments?	To identify cells that have successfully incorporated the transgene.
What type of selectable marker is commonly used?	AN antibiotic resistance gene
What is genome editing?	The precise modification of an organism's existing DNA.
Which technology is most commonly used for genome editing?	CRISPR-Cas9
Does genome editing always involve inserting foreign DNA?	No
What does CRISPR-Cas9 do?	It creates targeted DNA breaks that allow specific genetic changes to be made.
What is the major difference between transgenesis and genome editing?	Transgenesis introduces foreign DNA, whereas genome editing edits existing DNA
Which technology usually introduces a transgene?	Genetic modification (transgenesis)
Which technology may leave no foreign DNA in the final plant?	Genome editing
Which technology commonly uses Agrobacterium?	Transgenesis
Which technology commonly uses CRISPR-Cas9?	Genome editing
Why is genome editing considered more precise than transgenesis?	It targets specific DNA sequences for modification without necessarily adding foreign genes.
Why can genome editing be faster than traditional breeding?	Desired mutations can be introduced directly rather than waiting through multiple generations of crossing.
Why may genome editing be more publicly acceptable than transgenesis?	Some edited plants contain no foreign DNA and may resemble naturally occurring mutations.

Created by: Samara Hayes

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