Final Exam - Molecular Biology

Origin of replication

Site where replication begins.

Replicon

The total length of DNA replicated from one origin.

Prokaryotes: one origin
Eukaryotes: multiple origins

Five Steps of Replication Fork Assembly

1. Origin Recognition & Open Complex Formation

2. Helicase Loading

3. DNA Unwinding and Primer binding

4. Assembly of a Pol III holoenzyme complex

5. Coordination of helicase-polymerase

Replication Initiation in E. coli Is Controlled at Multiple (five) Steps

1. DNA Methylation & SeqA Sequestration

2. ATP/ADP Cycle of DnaA

3. Coupling with RNA Polymerase Activity

4. Titration of DnaA Binding Sites

5. Nutrient Status

Termination of DNA replication in E coli

Replication terminates opposite oriC.

Tus protein binds Ter sites → blocks DnaB helicase.

Polarity of Tus-Ter complex: blocks forks from nonpermissive direction and allows forks from permissive direction to pass.

End replication problem in linear chromosomes (eukaryotes) and solution (telomerase)

Linear chromosomes → lagging strand cannot fully replicate at the 3′ end. Removal of RNA primer leaves a gap with no 3′-OH for DNA polymerase. Leads to progressive shortening of chromosomes over cell divisions. Results in loss of genetic information and cellular dysfunction, aging, or tumor formation.

Telomerase binds 3′ end → aligns DNA with RNA template. Adds repeat unit. Repositions and repeats extension processively. DNA polymerase + primase complete lagging strand. Telomeres maintain stability, allow multiple divisions, and buffer against end loss.

DNA Polymerase III Core Structure and Function

Primary enzyme for E. coli chromosomal replication. The Pol III core is a heterotrimer composed of three subunits called α, ε, and θ.

Pol III Holoenzyme Organization and functions

3 β sliding clamps – ring-shaped, increase processivity

1 clamp loader (τ complex) – assembles β clamps onto the DNA

t subunits (3 copies)

Function: coordinates leading and lagging strand synthesis simultaneously, Fasten DNA synthesis speed.

The β Sliding Clamp – Speed & Processivity

β clamp = homodimer, ring encircling DNA, sixfold symmetry.

Binds Pol III core → converts from distributive to processive enzyme.

With B clamp: full circle in seconds, maintains tight attachment to DNA during processive synthesis.

Coordinated Protein Activities at the Replication Fork

Replication fork progression involves many proteins beyond Pol III core and β clamp.

These proteins:
- Unwind DNA (Helicase)
- Relieve supercoils (Topoisomerase)
- Prime DNA synthesis (Primase)
- Process RNA primers (Pol I, RNase H, Ligase)
- Stabilize ssDNA (SSB)

Types of DNA mutations

Point mutation (single base pair)

Insertion or deletion (more than one base pair)

Chromosomal rearrangement (many genes)

Transition vs transversion

Transition converts a purine-pyrimidine base pair to the other purine-pyrimidine pair, or pyrimidine-purine to pyrimidine-purine.

Transversion converts a purine-pyrimidine base pair to a pyrimidine-purine pair, or vice versa.

Point mutations types

Silent mutation = change that produces a codon for the same amino acid.

Missense mutation = change that results in different amino acid.

Nonsense mutation = changes the nucleotide sequence, instead of encoding an amino acid, the triplet functions as a stop codon, terminating the translation process.

Two-steps of Point mutations

First, an incorrect nucleotide is incorporated by a DNA polymerase. Then, incorporates the mismatch into a fully base-paired duplex DNA.

Insertion and deletion

Insertion and deletion mutations are the addition and removal of nucleotides in a DNA sequence.
Insertions and deletions that are not multiples of three shift a gene’s reading frame, resulting in a truncated protein.

Causes of mutations: DNA Alterations That Lead to Mutations

Hydrolysis, Oxidation, Alkylation, DNA-Damaging Agents, Radiation

Types of DNA Repair and mechanisms

Direct repair (Direct reversal of damage), Excision repair (Excision of damaged
region, followed by precise replacement), Recombination repair (strand break repair), Damage bypass

Mismatch repair - Strand Discrimination in E. coli

How the system knows which strand to fix: Dam methylase methylates the parental strand at 5′-GATC-3′ sequences. After replication → new strand is unmethylated for a
short time (hemimethylated state).

Base excision repair - Repairs Subtle Alterations in Nucleotide Bases

Purpose: Repairs small, non-helix-distorting base lesions (e.g., deamination, oxidation, alkylation).

Main Function: Removes a single damaged base; also processes single-strand breaks that require end “cleaning.”

Nucleotide excision repair - Repair Removes Bulky Damaged Bases

Corrects any damage that distorts the DNA molecule.

In all organisms, NER involves the following steps:
1. Damage recognition.
2. Binding of a multi-protein complex at the damaged site.
3. Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5’ and 3’ sides.
4. Removal of the damage-containing oligonucleotide from between the two nicks.
5. Filling in of the resulting gap by a DNA polymerase.
6. Ligation.

Main classes of RNAs

mRNA (messenger), tRNA (transfer), rRNA (ribosomal), other RNAs (miRNA, snRNA, snoRNA)

Transcription direction, phases, and differences from DNA replication

Direction: RNA synthesized 5′→3′, copying the 3′→5′ DNA strand.

Phases: Initiation, Elongation, Termination.

Differences: Transcribes specific genes, not whole genome, no primer required, only one DNA strand serves as template.

The Chemistry of RNA Synthesis

Requires: DNA template, Mg²⁺, and all 4 rNTPs (ATP, GTP, UTP, CTP).

Bacterial RNA polymerase structure and function of sigma factor

Structure: core enzyme, holoenzyme (core + sigma factor)

Function of sigma factor: directs polymerase to specific factors, determines promotor specificity - controls gene expression.

Eukaryotic RNA Polymerase types and functions

Pol I - 

Pol II - mRNA synthesis

Pol III - 

Bacterial promoters

Consensus sequences: sequences that possess considerable similarity.

Sigma Factors Specify Polymerase Binding to Particular Promoters

Consensus sequences in promoter

In bacterial promoters, consensus sequences are found upstream of start site.

Rho-dependent and Rho-independent termination

Rho-dependent: uses rho factor

Rho-independent: does not use rho factor

Eukaryotic Transcription Is Similar to Bacterial Transcription but Has Some Important Differences

Bacterial cells = holoenzyme (RNA polymerase core enzyme + the sigma factor) recognize and binds directly to sequences in the promoter.
Eukaryotic cells = promoter recognition is carried out by accessory proteins that bind to promoter and then recruit RNA polymerase (I, II or III).

Core promoters and regulatory promoters

Core: basal transcription apparatus, transcriptional activator proteins, and RNA polymerase II-mRNA synthesis.

Regulatory: variety of different consensus sequences may be found in the regulatory promoters.

Initiation: TATA box

Binding of the TFIID transcription factor to the TATA box.

Elongation

The DNA double helix enters RNA polymerase II through a cleft in the enzyme and unwinds.

The DNA–RNA duplex is bent at a right angle, which positions the 3' end of the RNA at the active site of the enzyme.

At the active site, new nucleotides are added to the 3' end of the growing RNA molecule.

Termination

Transcription by RNA polymerase II is terminated when an exonuclease enzyme.

Attaches to the cleaved 5' end of the RNA.

Moves down the RNA.

Reaches the polymerase enzyme.

Exons and introns in eukaryotes

Both are transcribed into RNA, but introns are later removed by RNA processing.

Introns are common in eukaryotic genes but uncommon in bacterial genes.

Types of RNA Processing in Eukaryotes

5′ Capping – modified guanosine added to protect RNA.
Splicing – removal of introns, joining of exons.
Polyadenylation – addition of a 3′ poly(A) tail.
RNA Editing – alteration of RNA sequence post-transcription.

Eukaryotes vs Bacteria in RNA splicing

5’-Capping

Carried out by RNA polymerase II.

Added early by RNA polymerase II–associated enzymes.
Structure: 7-methylguanosine (7-meG) linked by a 5′–5′ triphosphate bridge.
Functions: Protects mRNA from 5′→3′ exonucleases.
Required for ribosome binding via the cap-binding complex (CBC).
Aids in nuclear export.
Capping enzyme: Guanylyltransferase + methylation (from S-adenosylmethionine-SAM).

Experiment to support 5’-Capping

Aaron Shatkin’s reovirus mRNA study (1970s)

Capped mRNAs → translated
Uncapped mRNAs → no translation

Conclusion: 5′ cap is essential for translation initiation.

3’-Poly(A): process and functions

Process: Pol II extends the transcript beyond the site where the poly(A) tail is to be added.
The transcript is cleaved at the poly(A) addition site by an endonuclease component of a large enzyme complex, again associated with the CTD of Pol II.
The poly(A) tail is synthesized by polyadenylate polymerase (PAP).

Functions: Protects from 3′→5′ exonuclease degradation, interacts with poly(A)-binding proteins, enhances translation efficiency and mRNA stability.

Spliceosome and consensus sequences for splicing

Spliceosome: RNA-protein complex responsible for most splicing.

Consensus sequence: 5’ consensus sequence (splice site) and 3’ consensus sequence.

Two steps of pre-mRNA splicing processing

1. The 5' end of the intron is cleaved from the upstream exon and attached to the branch point to form a lariat.

2. The 3' end of the intron is cleaved from the downstream exon, and the ends of the two exons are spliced together.

Pre-mRNA Processing

RNA splicing takes place within the spliceosome

Alternative splicing and multiple 3’ cleavage sites

Enables exons to be spliced together in different combinations to yield mRNAs that encode different proteins.

RNA editing is carried out by

guide RNAs

Crick’s Adaptor Hypothesis

Explain how RNA directs protein synthesis.

Suggested the existence of an adaptor molecule that: recognizes specific codons in mRNA, carries a specific amino acid.

Structure of tRNA

Small, single-stranded RNA with a 3’-CCA terminus that folds into a cloverleaf - L-shaped structure.

Four major arms: amino acid arm, anticodon arm, D arm, TvC arm.

Aminoacyl-tRNA synthetase: types and functions

Enzymes responsible for “charging” tRNAs with their correct amino acids.

Degeneracy of the Genetic Code

64 possible codons (3 stop codons).

Degenerate code: multiple codons specify the same amino acid.

Advantages: provides mutational tolerance and reduces harmful effects of point mutations.

Key point: the genetic code is redundant but not ambiguous.

Results of wobble base pairing

One tRNA can recognize multiple codons.

Start codon and stop codons

Start: AUG (methionine)

Stop: UAA, UAG, UGA

Mutation Resistance of the Genetic Code (ref to Ch12)

The genetic code is highly error-tolerant.

Wobble and redundancy buffer translation errors. The code’s degeneracy and organization protect against translation and mutation errors.

Suppressor tRNAs function?

Nonsense mutations create premature stop codons → truncated proteins.

Suppressor tRNAs arise via mutations in anticodon sequence → allow amino acid insertion at stop codon.

Usually found in minor tRNA genes - avoids global suppression.

Suppression efficiency controlled to avoid misreading genuine stop codons. Suppressor tRNAs can “rescue” nonsense mutations, allowing partial restoration of protein function.

The Rules of the Genetic Code

The Genetic Code Is Nonoverlapping
There Are No Gaps in the Genetic Code
The Genetic Code Is Read in Triplets
Protein Synthesis Is Linear

Ribosomes and their compositions

Macromolecular machines that synthesize proteins. Present in cytosol, mitochondria, and chloroplasts.

Composed of rRNA (60%) and ribosomal proteins (40%).

Five steps of translation

1. The binding of amino acids to transfer RNAs

2. The initiation of translation

3. Elongation

4. Termination

5. Protein folding

Subunit Association During Translation

Small subunit binds mRNA and initiator tRNA first.

Large subunit joins → active 70S or 80S ribosome.

Reads mRNA 5′→3′ direction.

Subunits dissociate after protein release.

Recycled for next translation.

Ribosomal Binding Sites

A site: aminoacyl-tRNA entry

P site: peptidyl-tRNA (growing chain) (first tRNA locates here)

E site: exit for uncharged tRNA

Key Four stages of translation initiation

1. Recruitment of small ribosomal subunit to mRNA.

2. Identification of start codon (AUG).

3. Association of charged initiator tRNA with mRNA.

4. Recruitment of large subunit to form active ribosome.

How does rRNA bind to mRNA in bacteria? (SD sequence)

Small subunit binds mRNA and initiator tRNA first.

Initiation factors and GTP

The initiation of translation requires several initiation factors and GTP.

Eukaryotic mRNAs Recruit Small Subunit Indirectly

In bacterial cells, sequences in 16S rRNA of the small ribosomal subunit bind to the Shine–Dalgarno sequence in mRNA.

Eukaryotes: No analogous consensus sequence exists in eukaryotic mRNA. Instead, the cap at the 5 ′ end of eukaryotic mRNA plays a critical role in the initiation of translation.

Eukaryotic initiation requires at least 12 initiation factors.

The Initiation of Translation In Eukaryotes

The Kozak sequence in eukaryotic cells facilitates the identification of the start codon.

Purine before AUG, G after AUG enhances initiation.

Poly(A) tail at 3’--- facilitates translation.

Initiation in Bacterial Cells Requires Three Ifs (which one binds to GTP?)

IF 2

Initiation in eukaryotic Cells Requires more eIfs (which one binds to GTP?)

EIF 2

Components Required for Elongation

Ribosome: 70S (30S + 50S subunits).

tRNAs: Carry amino acids to mRNA codons.

Elongation factors.

Energy source: GTP hydrolysis powers movement and proofreading.

Three steps for elongation

1. Aminoacyl-tRNA Binding

2. Peptide Bond Formation

3. Translocation

Initiation of translation in bacteria and eukaryotes

Termination of translation

Stop codons: UAA, UAG, UGA.

eRF1 recognizes all stop codons.

Peptidyl transferase transfers chain to H₂O → polypeptide released.

Importance and Levels of Regulation

Ensures efficient use of energy and resources.

Allows adaptability to new conditions (nutrients, stress, development).

Prevents disease — e.g., mutations in regulatory genes cause cancer.

Regulation of Transcription Initiation

Highly efficient because it determines whether transcription begins at all, before the cell invests energy in mRNA synthesis and protein production.

Housekeeping genes and regulated genes

Housekeeping: constitutively expressed; steady levels for essential functions.
Regulated: expression varies with environmental or cellular needs.

Activation vs repression

Activation = increased expression.
Repression = decreased expression.

Positive regulation vs negative regulation

Positive regulation: activator binding increases transcription.
Negative regulation: repressor binding decreases transcription.

Enhancers and insulators: functions

Enhancers are distant regulatory sites in eukaryotes.

Insulators ensure that each enhancer or repressor acts only on its target gene.

Operon structure

Promoter + additional sequences that control transcription (operator) + structural genes

Regulator Gene

DNA sequence–encoding products that affect the operon function but are not part of the operon; with its own promoter!

Negative inducible operons: features, example (lac operon), and how the regulation works

The control at the operator site is negative. Molecule binding is to the operator, inhibiting transcription. Such operons are usually off and need to be turned on, so the transcription is inducible.

Inducer: small molecule that turns on the transcription.

Lac Operon: lactose metabolism

Negative repressible operons: features, example (trp operon), and how the regulation works

The control at the operator site is negative. But such transcription is usually on and
needs to be turned off, so the transcription is repressible.

Corepressor: a small molecule that binds to the repressor and makes it capable of binding to the operator to turn off transcription.

Trp operon: tryptophan synthesis

Positive control and catabolite repression: use glucose-lac regulation as an example

Catabolite repression: using glucose when available and repressing the metabolite of other sugars.

CAP binds to lac operon when glucose level is low.

Integration of two signals (glucose + lactose) in lac operon regulation

Lactose: absent, absent, present, present

Glucose: high, low, high, low

Operon: off, off, mostly off, on

Reason: repressor bound and no lactose to remove it, repressor bound, CAP not activated (low cAMP), repressor inactive + CAP helps RNA polymerase bind

Comparison: Inducible vs. Repressible Operons

Purpose: I = controls catabolic pathways; R = controls anabolic pathways

Regulation mechanism: I = inducer binds repressor, repressor releases operator, transcription proceeds; R = corepressor binds repressor, repressor binds operator, transcription stops.

Goal of regulation: I = prevent unnecessary enzyme production when substrate is absent; R = prevent overproduction of an end product when it is abundant.

Difference of gene regulation between bacteria and eukaryotes

Each structural gene has its own promoter and is transcribed separately.

DNA must unwind from the histone proteins before transcription.

Transcription and translation are separated in time and space.

Three processes affect gene regulation by altering chromatin structure?

Chromatin remodeling, modification of histone proteins, DNA methylation

Transcription activator proteins have two distinct functions

1. Binding to a consensus sequence in a regulatory
   promoter or enhancer (DNA-binding motifs)

2. Interacting with other components of the basal
   transcription apparatus

What are silencers, enhancers and insulators?

Silencer: DNA sequence with an inhibitory effect on the transcription of distant genes.

Enhancer: DNA sequence stimulating transcription a distance away from promoter.

Insulator: DNA sequence that blocks or insulates the effect of enhancers.

How does insulator help gene regulation?

Insulators help create neighborhoods of gene regulation.

Degradation of RNA

RNA degraded by ribonucleases.

RNA interference by small interfering RNAs and microRNAs (compare the mechanism)

Dicer and RISC: RNA-induced silencing complex.

siRNAs target a single mRNA with perfect match leading to direct cleavage, while miRNAs are from hairpin RNA, often target multiple mRNAs with partial match.

Comparison of gene control in bacteria and eukaryotes

Levels of regulation: (B) primarily transcription; (E) many levels

Cascades of gene regulation: (B) present, (E) present

DNA-binding proteins: (B) important, (E) important

Role of chromatin structure: (B) absent, (E) important

Presence of operons: (B) common, (E) uncommon

Negative and positive control: (B) present, (E) present

Initiation of transcription: (B) relatively simple, (E) relatively complex

Enhancers: (B) less common, (E) more common

Transcription and translation: (B) occur simultaneously, (E) occur separately

Regulation by siRNAs and miRNAs: (B) absent, (E) common

Principles of PCR (features)

PCR amplification is achieved by using oligonucleotide primers.

cDNA

(complementary DNA) is made from mature mRNA, so it contains no introns.

Plasmid features

Plasmids: circular DNA molecules from bacteria.

Insert foreign DNA into plasmid using restriction enzymes.

Restriction enzymes/sites

Enzymes that recognize specific nucleotide sequences in DNA and make double-stranded cuts at those sequences (called restriction sites).

Sticky ends and blunt ends

Sticky ends: fragments with short, single-stranded overhanging ends.

Blunt ends: even-length ends from both single strands.

Principle of LacZ gene to screen for recombinant plasmids

Foreign DNA is inserted into lacZ gene. Bacteria that are LacZ- are transformed by the plasmid. Bacteria without a plasmid will not grow.