Cell Adhesion, ECM, and DNA Replication: Key Molecular Mechanisms

ECM = protein fibers embedded in a gel of glycosaminoglycans (GAGs)

Extracellular Matrix (ECM)

GAGs + proteins → proteoglycans

Proteoglycans

Link ECM components and cells to ECM (e.g., collagen, proteoglycans)

Adhesion proteins

Integral membrane proteins made of two subunits that bind specific amino acid sequences in ECM

Integrins

Stable adhesion junctions

Desmosomes

Involved in cell-cell adhesion

Selectins/Integrins

Formed by two connexons (each made of 6 subunits) allowing direct cytoplasmic exchange between cells

Gap Junctions

Stabilize DNA polymerase during replication

Clamp-loading & sliding-clamp proteins

Unwind DNA during replication

Helicases

Stabilize single-stranded DNA

DNA-binding proteins

Relieve supercoiling tension during DNA replication

Topoisomerases

Multi-enzyme complex coordinating DNA replication

Replisome

Loops to allow both polymerases to move in the same direction during DNA replication

Lagging strand

Extends telomeres and is active in embryonic stem cells; mutations linked to cancer

Telomerase

σ factor binds -10 and -35 promoter regions → closed complex; DNA unwinds (12-14 bp) → open complex

Bacterial Transcription Initiation

Begins RNA synthesis after σ factor dissociates after ~10 nucleotides

RNA polymerase

Requires 5 general transcription factors to form preinitiation complex with RNA polymerase II

Eukaryotic Transcription Initiation

Recruits RNA endonuclease for termination in eukaryotic transcription

CTD phosphorylation

Aids export, prevents degradation, and assists ribosome binding in mRNA processing

5′ Cap

Prevents degradation and regulates translation in mRNA processing

Poly-A Tail

Acetylation → chromatin decondensation → ↑ transcription

Histone Modification

CpG methylation → transcriptional repression

DNA Methylation

Directs ribosome to ER during cotranslation and ER targeting

Signal Sequence

Triggered by excess unfolded proteins, increases chaperones and proteasomes, limits new protein entry; failure leads to apoptosis

Unfolded Protein Response (UPR)

COPII: ER → Golgi; COPI: Golgi → ER (retrograde); Clathrin: Golgi ↔ endosomes/lysosomes/plasma membrane

Coated Vesicles

v-SNARE (vesicle) + t-SNARE (target) ensure specificity in vesicle targeting and fusion

SNAREs

Core protein + glycosaminoglycan (GAG) chains

Proteoglycan structure

Chondroitin sulfate, heparan sulfate

Examples of GAGs

Meshwork of lamin proteins lining the inner nuclear membrane

Nuclear Lamina

Provides structural support and organizes chromatin

Function of Nuclear Lamina

Transmembrane receptors that link ECM to cytoskeleton

Integrins

Actin filaments via focal adhesions

Intracellular partners of Integrins

Mediate transient cell-cell interactions (e.g., leukocyte rolling)

Selectins

Inflammation and immune response

Key process involving Selectins

Dense plaques with cadherins (desmoglein, desmocollin)

Desmosomes

Anchor intermediate filaments, provide mechanical strength

Function of Desmosomes

Barrier to paracellular transport

Tight Junctions

Direct ion/molecule exchange

Gap Junctions

Channels allowing passage of ions, small molecules (<1 kDa)

Connexons

Synthesizes RNA primers

Primase

Extends primers

DNA polymerase

Seals Okazaki fragments

DNA ligase

Due to antiparallel strand orientation

Discontinuous synthesis

Begins at specific DNA sequences

Initiation

Primase starts without pre-existing strand

De novo synthesis

Sliding clamp that tethers DNA polymerase to DNA

PCNA

Clamp loader complex positions PCNA on DNA

Loading

Stabilize single-stranded DNA, prevent reannealing

SSBs

Relieve supercoiling ahead of replication fork

Topoisomerases

Ensures correct nucleotide incorporation

Base-pair selection

3′→5′ exonuclease activity removes mismatches

Proofreading

Extends telomeres using RNA template

Telomerase Function

Prevents chromosome shortening, maintains stability

Role of Telomerase

Guides RNA polymerase to promoter (-10, -35 regions)

σ factor

DNA unwound, transcription begins

Open complex

Assemble at promoter

General transcription factors

Joins complex to begin transcription

RNA polymerase II

Triggers cleavage of pre-mRNA

Polyadenylation signal

Degrades downstream RNA, dislodges polymerase

Exonuclease

Bridges transcription factors and RNA pol II

Mediator Complex

Aids nuclear export, protects from degradation, promotes translation

5′ Cap

Stabilizes mRNA, regulates translation efficiency

Poly-A Tail

Induces operon by inactivating repressor

Lac Operon

Inhibits operon via low cAMP

Glucose

Activator that binds when cAMP is high

CAP

Increase transcription rate

Enhancers

Coordinates transcription and RNA processing

RNA Pol II CTD

Pause transcription

Negative regulators

Releases paused polymerase via phosphorylation

P-TEFb

Methylation represses transcription

DNA Methylation

Methylation represses transcription and condenses chromatin

CpG islands

Polycistronic, no cap/tail

Prokaryotic mRNA

Monocistronic, 5′ cap, poly-A tail

Eukaryotic mRNA

Extensive (splicing, capping)

Processing of Eukaryotic mRNA

Coupled with transcription

Translation in Prokaryotes

Separate from transcription

Translation in Eukaryotes

Small ribosomal subunit, mRNA, fMet-tRNA, initiation factors

Initiation in Prokaryotes - Key molecules

AUG recognized by fMet-tRNA

Start codon

Directs ribosome to ER

Signal sequence

Binds signal, pauses translation

SRP

Docking site on ER membrane

SRP receptor

Accumulation of misfolded proteins

Unfolded Protein Response (UPR) - Triggers

↑ chaperones, ↑ proteasomes, ↓ new protein entry

Unfolded Protein Response (UPR) - Responses

Apoptosis if unresolved

Unfolded Protein Response (UPR) - Outcome

Guides insertion into membrane

Cleavable signal

Opens to allow passage

Translocon

Pinches off vesicle from membrane

Dynamin

Endocytosis and intracellular transport

Importance of Dynamin

ER → Golgi, Forward transport

COPII

Golgi → ER, Retrograde transport

COPI

Golgi ↔ PM/lysosomes, Bidirectional sorting

Clathrin

Guide vesicles to correct membrane

Rab proteins

v-SNARE (vesicle) + t-SNARE (target) ensure specificity

SNAREs

SNAREs form coiled coil, pull membranes together

Fusion of Vesicles