Part 2 108A Final (chapter 11, 25, 26, 27)

Integral membrane proteins

Span the lipid bilayer and interact with the hydrophobic core.

They are often alpha-helical or beta barrel structures.

Peripheral membrane proteins

Attach to membrane surface via non-covalent interactions or to integral proteins

Do NOT interact with hydrophobic core

These are on the outside

Hydropathy plot

Graph of hydrophobicity vs amino acid position; predicts membrane spanning segment

• X axis represents the amino acid sequences

• Y axis represents hydrophobicity (positive values indicate hydrophobic regions and negative values indicate hydrophilic regions)

By analyzing peaks and valleys, you can identify potential membrane spanning domains (such as if the graph shows a region with hydrophobic and hydrophilic areas we can interpret this as a possible transmembrane protein)

Membrane fusion

Process of two lipid bilayers merging to become one

• Two separate bilayers merge into a continuous bilayer

• Ex: Fusion of synaptic vesicles with the cell membrane in nerve cells release neurotransmitters to transmit signals or viral entry into host cells

v-SNARE

Proteins on the vesicle that mediate vesicle fusion

• ON the vesicle membrane

• Cytoplasmic face of neurotransmitter vesicle

t-SNARE

Proteins on the vesicle that mediate vesicle fusion

• ON the target molecule

• Contained in target molecule (located on the target molecule)

What do v-SNARE and t-SNARE do?

These proteins interact to form a stable trans-SNARE complex, bringing the vesicles and target membrane close together and leading to membrane fusion

Facilitated diffusion

Passive transport via membrane proteins (channels or carriers, NO ATP)

SERCA Pump

A P-type ATPase that pumps Ca²⁺ sarcoplasmic reticulum using ATP

• its primary function is to transport Ca²⁺ ions from the cytoplasm into the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) using energy from ATP hydrolysis

• This helps maintain low Calcium concentrations in the cytoplasm, which is essential for various cellular processes

V-Type ATPases

V type stands for vacuoles that hydrolyze energy (ATP) to create ion gradients (vicky and licky make energy gradients)

Vacuolar

Pumps protons into organelles (lysosomes) using ATP

Located in various intracellular membranes like lysosomes and vacuoles and they primarily hydrolyze ATP to create ion gradients for cellular processes

F Type ATPase

• F type, First type best type, found in mito and chloro - obvi best parts of the cell, synthesizing the energy of the cell

They are primarily found in mitochondria and chloroplasts, where they synthesize ATP using electrochemical proton gradients generated by respiration or photosynthesis

Na+/glucose transporter (sodium and glucose transporter) (SGLT)

A protein that uses the electrochemical gradient of sodium ions to transport glucose across cell membranes

Secondary active transport (does not directly use ATP but relies on the movement of Na+ down their concentration gradient to power the movement of glucose against its concentration gradient)

Approximate value of proteins

30%-70%

Vary based on specific membranes function and location within the cell

Example: The inner mitochondrial membrane involved in ATP production is about 75% protein by mass while the myelin membrane contains less than 25% protein.

Approximate value of Phospholipids

7% to 40%

• Structural component of cell membranes

Approximate value of Sterols (cholesterol)

0%-25%

Cholesterol is the major sterol in animal cell membranes comprising about 30% of the lipid bilayer on average

What are photoreceptor disc membranes characterized by?

A high concentration of rhodopsin (over 90% of the membrane protein)

Lipid-Anchored protein

Covalently attached to lipid molecules embedded in the membrane

What are two common features about the amino acid composition of transmembrane proteins?

1. Abundance of hydrophobic residues (Leu, Val, Ile) in the transmembrane region - crucial for their integration and stability within the lipid bilayer which is a hydrophobic environment formed by the fatty acid tails of membrane lipids

2. Tyr and Trp residues and lipid water interface- their amphipathic nature helps anchor the protein at the boundary between the hydrophobic membrane interior and aqueous environment

How many Amino acids span the membrane in an alpha helix?

Around 20 non-polar amino acids span around 30 angstroms hydrophobic core of a bilayer

An alpha helix rises 1.5 A per residue, so 30A/1.5A = 20 residues

Each alpha helical turn is 3.6 residues, spanning the membrane takes about 5-6 turns

Transcriptome

The complete set of RNA transcripts (including mRNA, tRNA, rRNA, and other non-coding RNAs)

What does it mean that membrane lipids are amphipathic?

They have both hydrophilic (polar) head groups and hydrophobic (nonpolar) fatty acid tails

Why do lipids spontaneously assemble in water.

To minimize free energy by segregating hydrophilic heads toward water and hydrophobic tails away from water

What is a micelle and when is it formed

A spherical lipid structure formed by single-tailed lipids (detergents) with hydrophobic tails inward and polar heads outward

What is a lipid bilayer

Two leaflets of lipids forming a planar or curved sheet that encloses an aqueous compartment (vesicles or lipsomes)

Approximately how thick is a lipid bilayer?

About 3 nm (30 A) thick

Are the inner and outer leaflets of a lipid bilayer identical

No they are asymmetrical meaning they have different lipid compositions

What does membrane fluidity mean?

Lipids and proteins can move laterally within the membrane, contributing to its dynamic nature.

What is the functional purpose of the lipid bilayer’s barrier?

It provides selective permeability, separating the cell from its environment and maintaining distinct internal compartments

What are the key criteria that must be met for membrane fusion to occur

1. Membrane continuity must be preserved

2. Membranes must recognize each other

3. Close proximity with water removal (local dehydration)

4. Fusion proteins (eg SNAREs) must mediate the process

What structural changes happen during membrane fusion?

1. Increase in local membrane curvature

2. Disruption of bilayer at contact point
   Hemifusion (outer leaflets fuse first)

3. Full fusion creates a single continuous bilayer

What roles do v-SNAREs and t-SNAREs play in membrane fusion?

They interact to bring membranes close together, overcome energy barriers, and facilitate bilayer distortion for fusion

What additional requirements are often needed for membrane fusion during endocytosis or secretion?

A trigger or signal (calcium or ligand binding) and energy input (ATP or GTP hydrolysis)

What type of transport is Glut1?

Facilitated diffusion of glucose; passive transport follows the concentration gradient

• Transports glucose into RBCs

• Specific for glucose over sugars

• Glut1 is a type III integral membrane protein, meaning it spans the cell membrane multiple times with both its N and C termini inside the cell

How would a membrane adjust to become more liquid-disordered until certain temperature changes (more fluid)

Increase unsaturated fatty acids (kinks/double bonds)

Shorter fatty acid chains

Higher temperature

How would a membrane adjust to become more liquid-ordered until certain temperature changes (less fluid)

• Increase cholesterol

• More saturated fatty acids

• Lower temperature

What is the equation for delta G of transport for an uncharged solute?

What is the equation for delta G of transport for an charged solute?

Same equation for an uncharged solute plus Z (charge) times F (faradays) times membrane potential

What is the primary function of P-type ATPases

To actively transport cations (Ca2+, Na+) against their concentration gradients using ATP through reversible phosphorylation of an aspartate residue

What are the three cytoplasmic domains of P-type ATPase and their functions?

N Domain: Binds ATP and Mg2+, phosphorylates Asp in P domain

P Domain: contains the Asp residue that gets phosphorylated

A domain: acts as activator, removes the phosphate to reset the cycle

How does the mechanism of P-type ATPases drive ion transport?

1. ATP binds to the N domain

2. Phosphate is transferred to Asp in the P domain

3. Conformation change (E1—> E2) moves the ion across membrane

4. A domain removes phosphate, resetting the protein

What is the SERCA pump and how does it function

A P-type ATPase in the sarcoplasmic reticulum of muscle cells

Pumps 2 Ca2+ ions into the SR lumen per ATP hydrolyzed

Phosphorylation shifts it from E1 (high Ca2+ affinity) to E2 (low Ca2+ affinity)

Essential for muscle relaxation

Okazaki Fragment

Short DNA segments (1000-2000nt in bacteria, 150-200 nt in eukaryotes) synthesized discontinuously on the lagging strand during DNA replication. Later joined by DNA ligase

DNA Pol I

Not the main polymerase for replication. It has low processivity and functions mainly in the removal and replacement of RNA primers, as well as in repair and cleanup

DNA Poly III - repliaction

The main replicative polymerase in E.Coli. It adds nucleotides during elongation on both the leading and lagging strands.

RNaseH1

Type of replication enzyme

-Specialized nuclease that degrades RNA in RNA-DNA hybdris

• Not typical, in case something goes wrong

Topoisomerase

An enzyme that relieves topological stress ahead of replication forks by cutting and rejoining DNA strands. Topoizomerase IV resolves catenanes during termination

• replication

Ligase

Seals nicks in the DNA backbone after primers are replaced, forming a continuous strand 

Primase

Enzyme that synthesizes short RNA primers (10-60 nt) to initiative DNA synthesis in E.Coli, it's known as DnaG, and works with helicase.

Catenane

Two interlinked circular DNA molecules formed after replication of circular chromosomes. Separated by topoisomerase IV.

What does bidirectional replication mean?

Replication proceeds in both directions from a single origin (oriC in E.Coli) creating two replication forks moving away from the origin 

• How does DNA proofreading occur?

DNA polymerase (like DNA pol III) have 3’--> 5’ exonuclease activity 

• If the wrong nucleotide is added it alters the geometry 

• The polymerase pauses and removes the incorrect base 

• This improves accuracy 100-1000 fold 

α (alpha)

Polymerase activity

allen loves pollys ass 

β (beta)

Sliding clamp for processivity 

You beta slide that clamp over

ε (epsilon)

3’ to 5’ exonuclease (proofreading) 

looks like an e for epsilon or exonuclease (always works 3—>5)

θ (theta)

stabilizes ε, part of core polymerase

stabilizes the exonuclease

• Theres an e in theta, its meant to be

• theta and episolon love eachother and theta wants to stabilize epsilon and become part of the core polymerase

• Stabilizes the exonucease

τ (tau)

links multiple core polymerases 

• Alpha Taus link with multiple people

γ, δ, δ′

 clamp loader complex

• gamma delta delta primes have a clamp loading complex

• meaning they really like to clamp

χ, ψ

bind to clamp loader 

• The ψ (psi) subunit joins the minimal complex by interacting with one of the γ subunits, forming a six-subunit complex (γ3δδ′ψ).

• The χ (chi) subunit then binds to the ψ subunit to form the seven-subunit clamp loader (γ3δδ′χψ). 

Chi and psi bind to the clamp loader to make it a 7 subunit clamp load

Chi Psis love a good seven man

RNA polymerase

Enzyme that synthesizes RNA from a DNA template. It adds ribonucleotides to the 3’-OH end in the 5’ to 3’ direction using ATP, UTP, and CTP and requires Mg²⁺

rho-dependent termination

In E.Coli this uses the rho protein, an ATP-depedent RNA-DNA helicase. Rho binds to CA rich sequence (rut site) and moves along the RNA to unwind the RNA-DNA duplex and release the transcrit

rho-independent termination:

Occurs via a self complementary region in the RNA that forms a hairpin loop followed by a string of U residues. This structure causes RNA polymerase to pause and release the RNA. 

Intron

A non coding sequence in a eukaryotic primary mRNA transcrip that is removed during splicing 

Exon

A coding region of an mRNA transcript that remains after splicing and is translated into protein

TATA box

A promoter sequences (TATA(A/T)A(A/T)(A/G) found 30 bp upstream of the transcription start site in many Pol II genes. It helps position RNA polymerase for initiation 

DNA Pol II

responsible for the synthesis of mRNAs and many ncRNAs 

What are the correct order of events for transcription?

1. Initiation- RNA polymerase binds to promoter (TATA box)

2. Promoter clearance- enzyme begins RNA synthesis and escapes promoter 

3. Elongation - RNA is synthesized in the 5’ to 3’ direction but adds bp 3—>5

4. Termination- RNA transcript is released and polymerase dissociates 

Prokaryotic Initiation

RNA pol holoenzyme (core + σ factor) binds to promoter sequences (eg, -35 TTGACA, -10 TATAAT) 

• No primer is needed 

• σ70 helps recognize promoter, dissociates as elongation begins 

Prokaryotic Elongation

• RNA pol synthesizes RNA complementary to template strand 

• DNA unwinds, forming a transcription bubble 

• NusA replaces σ and aids elongation 

Prokaryotic Termination

• Rho-independent: hairpin + U-rich sequence causes release 

• Rho-dependent: Rho protein unwinds RNA-DNA duplex at rut site 

Eukaryotic Initiation

• Involves RNA polymerase II and transcription factors (TFs) 

• Common promoter motifs: TATA box (around -30), Inr (+1) 

• A pre-initiation complex forms with Pol II and TF’s 

• CTD (C-terminal domain) of Pol II is phosphorylated to begin elongation 

Eukaryotic Elongation

• RNA synthesizes in 5’ to 3’ direction 

• DNA reanneals behind the transcription bubble 

Eukaryotic Termination

Pol II transcribes past the cleavage site 

RNA is cleaved and Poly(A) tail is added

Polyadenylation signals, no Rho

5’ capping - mRNA processing

1. Adds 7-methylguanosine cap via 5’,5’ triphosphate linkage 

2. Protects from degradation and helps ribosome binding 

Splicing - mRNA processing

Introns are removed, Exons are joined

Carried out by spliceosome or ribozymes

3’ Polyadenylation in mRNA processing

Poly(A)tail (80-250 A’s) added to polyadenylate polymerase

Protects from degradation and aids in export and translation

• This occurs in eukaryotic transcription because the new mRNA must be exported out to the cytoplasm, whereas the prokaryotic mRNA does not translation occurs simultaneously with transcription

RNA pol I

transcribes most rRNAs (eg 18S, 28S) 

RNA Pol II

Transcribes mRNAs and many ncRNAs

works in eukaryotes

RNA Pol III

Transcribes tRNAs, 5S rRNA, and other small RNA’s

What are the steps in rho-dependent termination in E. coli?

1. Rho protein binds to the rut site (C-rich region on RNA) 

2. It moves 5’ to 3’ using ATP hydrolysis 

3. Catches up with RNA polymerase stalled at a termination site 

4. Unwinds RNA-DNA hybrid → releases RNA transcript 

Codon

• A group of three nucleotides on an mRNA that encodes a specific amino acid 

• There are 64 codons (4³ combinations), sufficient for encoding 20 amino acids 

• The genetic code is degenerate: multiple codons code for the same amino acid  

• There are 3 stop codons and 1 initiation codon (AUG) 

Wobble hypothesis

• Explain the ability of some tRNAs to pair with more than one codon due to flexibility at the third position of the codon 

• First base of anticodon: 

  • A or C → specific pairing with U or G 

  • U or G → wobble pairing: U pairs with A or G; G pairs with U or C 

  • Inosine (I) in anticodon can pair A, U, or C 

• Only ~32 tRNAs are needed to read all codons due to wobble 

aminoacyl-tRNA synthetases: 

• Enzymes that attach the correct amino acid due to its corresponding tRNA (charging) 

• Highly specific: usually 1 synthetase per amino acid 

• Two steps: 

  • Formation to aminoacyl-AMP 

  • Transfer to tRNA’s 2’- OH (Class I) or 3’-OH (Class II) 

• Some have proofreading ability, Ile-tRNA synthetase removes mischarged Val 

EF-tu (Elongation Factor Tu)

• Binds GTP and delivers aminoacyl-tRNA to the A site during elongation 

• After GTP hydrolysis, EF-Tu-GDP is released 

IF-2

• Binds GTP and helps position fMet-tRNA at the P site during initiation 

• Facilitates binding of the 50S subunit after correct anticodon-codon pairing 

aminoacyl-tRNA

• A tRNA molecule charged with its specific amino acid 

• The amino acid is linked via ester bond to the tRNA’s 3’ terminal A residue (on CCA tail)

Peptidyl transferase

• Catalytic function of the ribosome (specifically the rRNA) 

• Forms peptide bonds between amino acids during elongation 

• Transfer peptide from tRNA in P site to the amino group of aminoacyl-tRNA in A site 

• No proteins are near the active site → Ribosome is a ribozyme

Bacterial ribosome big and small subunit

70S (30S + 50S) 

Eukaryotic ribosome

80S (40S + 60S) 

What is in ribosomes

rRNA and proteins —> 65% rRNA (catalytic core)

Ribosome is a ribozyme not a protein enzyme

What are the three binding sites in a Ribosome

• A site = accepts aminoacyl-tRMA 

• P site = Holds tRNA with growing polypeptide 

• E site: Exit site for uncharged tRNA 

Structure of tRNA

73-93 nucleotides in length 

2D: Cloverleaf; 3D: L- shaped 

Contains modified bases (methylated residues) 

What are the key structural features and functions of tRNA in protein synthesis?

• Size: 73–93 nucleotides, single-stranded RNA (ssRNA)

• Structure:

  • 5′ end: Guanylate (pG) residue

  • 3′ end: CCA sequence for amino acid attachment

  • Anticodon arm: Contains anticodon (wobble at 5′ position of anticodon)

  • Cloverleaf shape (2D), twisted L shape (3D)

  • D arm & TC arm: Help with ribosome recognition

• Function:

  • Adapter molecule: links specific amino acids to mRNA codons during translation

  • Ensures the correct amino acid is added to the growing polypeptide chain