MITOCHONDRIA

• Generate energy for the cell
• Have numerous metabolic functions
• Comprise mitochondrial proteins (most encoded by nuclear genes)
• Compartmental organization:
  i. Outer membrane ii. Intermembrane space iii. Inner membrane iv. Matrix

IMPORT INTO MITOCHONDRIA
• Prospective matrix protein unfolded in cytosol
• Presequence: signal sequence at N-terminus; positively charged residues
• TOM (translocase of outer membrane): import receptor and translocator
• TIM (translocase of inner membrane): import receptor and translocator
• Protein passes through TOM and enters intermembrane space; then passes through TIM to enter matrix

TOM-TIM alignment: research suggests that TIM tethers to the outer membrane and diffuses laterally until it comes into contact with the TOM/presequence complex

• Protein can enter matrix in energy-dependent manner (presequence cleaved by peptidase and chaperones assist in folding)
• Protein’s transmembrane domain can diffuse laterally out of TIM and embed in inner membrane

CLINICAL CORRELATION
Neurodegenerative disorders
• Often caused by defects in translocases
• Genetic disorder in TIM production results in deafness and dystonia

KEY MECHANISMS OF PROTEIN TRANSPORT

• Gated transport: energy-dependent (nuclear pores)
• Translocation across membranes: mediated by protein translocators (mitochondrion, post-translational and cotranslational import into ER or peroxisomes)
• Vesicular transport: proteins do not cross membranes (from ER to Golgi, through Golgi stacks or to plasma membrane)

PROTEIN TRANSLATION
• Synthesis of almost all proteins begins in the cytoplasm
• 3 possible outcomes depending on signal sequence (or lack thereof):
i. remain in cytosol ii. post-translational import into organelle (mitochondrion, peroxisome, nucleus, ER) iii. cotranslational import to ER

SIGNAL SEQUENCES
• Short amino acid sequences at the terminal end of proteins (address labels)
• 15-60 residues long
• Direct proteins to specific organelles
• Proteins without signal sequences remain in the cytosol

MEMBRANE TOPOLOGY
• Compartments are topologically equivalent if molecules can get from one to another without having to cross a membrane
• ER, Golgi, vesicle, perinuclear space in nuclear envelope are all topologically equivalent
• Mitochondrion, nucleus and peroxisomes are topologically distinct

• Removes misfolded proteins
• Regulates the amount of a protein in the cell at a time

Two major pathways

1. Ubiquitin-dependent pathway
2. Autophagy (targets old and worn-out organelles)

Proteolysis

Break down of proteins into smaller peptides or amino acids

Clinical Correlation

• Pathologic accumulation of misfolded proteins results in various diseases such as sickle cell anemia and certain neurodegenerative illnesses (Huntington’s disease, Alzheimer’s disease, and Creutzfeldt-Jacob disease)

PROTEASOME STRUCTURE – 26S Mammalian proteasome

1. 20S Subunit

• “Central cylinder”
• Comprised of four rings – two inner rings (seven beta subunits each) and two outer rings (seven alpha subunits each)
• Has protease activity

2. 19S Subunits

• “Cap” subunit
• ATPas sites – provide energy for protein degradation and unfolding by using ATP
• Ubiquitin binding sites – recognize proteins tagged for degradation

UBIQUITIN-DEPENDENT PROTEIN DEGRADATION PATHWAY

Protein tagging with ubiquitin

1. Ubiquitin added to cysteine side chain of the E1 protein (ubiquitin-activating enzyme)
2. E1 protein becomes bound to E2 (ubiquitin conjugating enzyme) and E3 protein complex (ubiquitin ligase)
3. Ubiquitin transferred to cysteine side chain on the E2 protein
4. Misfolded protein becomes bound to E3 protein
5. Ubiquitin transferred to lysine side chain on the misfolded protein
6. Repeat steps 1-5 to polyubiquitinate the misfolded protein – polyubiquitin chain is the signal for protein degradation in the proteasome

Proteasomal degradation of the tagged protein

1. Misfolded protein with polyubiquitin chain is recognized by 19S cap of the proteasome
2. Using ATP as an energy source, protein is unfolded, ubiquitin is released, and the protein is translocated into the 20S subunit to be degraded (unknown if unfolding or removal of ubiquitin happens first, but both must occur before translocation can happen)
3. When translocated protein reaches the proteolytic sites, it is cleaved and peptide fragments are released into the cytoplasm for further degradation

PROTEIN SYNTHESIS

Translation

Conversion of mRNA to protein

Codon

Group of three mRNA nucleotides that encode one amino acid

Ribosomal Structure

• 2 subunits each made of protein and RNA:

1. Small ribosomal subunit – has mRNA binding site
2. Large ribosomal subunit

• E-site (Exit site)
• P-site (Peptidyl-tRNA binding site)
• A-site (Aminoacyl-tRNA binding site)

PROTEIN SYNTHESIS STEPS

Initiation

• Initiator t-RNA (methionine) binds to small ribosomal subunit (with help from initiation factors)
• Small ribosomal subunit/initiator t-RNA complex binds mRNA (using 5′ cap to bind the 5′ end)
• Complex scans towards the 3′ end until it locates the start codon (AUG which sets the appropriate reading frame) and the large ribosomal subunit arrives

Elongation

• Current tRNA in the P-site
• New tRNA added to A-site
• Growing peptide chain added to amino acid bound to A-site tRNA
• Large ribosomal subunit shifts 3′ one codon
• Small ribosomal subunit follows – tRNA that was in P-site now in E-site and tRNA that was in A-site now in P-site
• E-site tRNA ejected and new A-site tRNA added

Termination

• When codon in A-site is stop codon (UAG, UAA, or UGA) a release factor is added to the A-site
• Release factor binding releases the peptide chain and causes ribosome to dissociate

POST-TRANSCRIPTIONAL MODIFICATIONS:

• Help with the export of the mRNA from the nucleus
• Protect the mRNA from degradation in the cytoplasm
• Help facilitate proper translation

3 TYPES OF MODIFICATIONS

• 5′ capping
• Poly-A tail
• Intron splicing

5′ capping

• Modified guanine nucleotide
• Added to 5′ end of RNA molecule
• Added during transcription

Poly-A tail

• Multiple adenines added at the end of the polyadenylation signal
• Added to the 3′ end of the RNA

Intron splicing

Intron

Sections of the RNA sequence that are found in between exons

• Introns are removed from the RNA by the spliceosome

Spliceosome

Group of small nuclear RNAs and proteins which join together to recognize and remove introns from transcribed RNA

• Products of intron splice are mature mRNA and circular intron

Alternative splicing allows for one gene to code for multiple, similar proteins

Incorrect splicing is often the cause of the disease Hutchinson-Gilford progeria syndrome

DIFFERENCES BETWEEN PROKARYOTIC AND EUKARYOTIC TRANSCRIPTION

Prokaryotes:

• Only one polymerase isoform
• Occurs in the cytoplasm (since no nucleus)
• RNA polymerase recognizes the promoter sequence
• RNA polymerase recognizes the terminator sequence
• No additional processing of mRNA

CONVERTS INFORMATION IN DNA TO RNA

• Protects original information
• Multiple RNA copies allow for more protein to be produced

DEFINITIONS

Transcription factors

Proteins which bind the promoter and recruit the RNA polymerase

Template strand

Strand of DNA in which the gene to be transcribed is located

THREE STEP PROCESS:

Initiation

• Promoter recognized by transcription factors
• RNA Polymerase II recruited by the transcription factors
• Transcription begins and continues 3′ to 5′ along template strand

Elongation

• Complementary RNA nucleotides added to the growing RNA strand (uracil instead of thymine in the RNA strand)

Termination

• Polyadenylation signal in the transcribed RNA signals proteins to cut it from the polymerase
• Newly formed RNA is called Pre-mRNA because it still needs to be processed
• RNA Polymerase eventually falls off the template strand

• Allows organisms to be more efficient with resources and to adapt to the environment
• Important for cell differentiation (which results in many different cell types)

STEPS REGULATION OCCURS

1. Transcriptional Control

• When and how often a gene is transcribed
• Only point where the cell will not synthesize unneeded intermediates, thereby wasting resources

2. RNA Processing Control

• Control of the splicing and processing of RNA transcripts

3. RNA Transport and Localization Control

• Cells select which mRNAs are exported to the cytoplasm and where they are localized to

4. Translational Control

• Control which mRNAs get translated into protein

5. mRNA Degradation Control

• Cells can destabilize specific mRNAs in the cytoplasm to limit protein production

6. Protein Activity Control

• Cells can selectively activate, inactivate, localize or degrade proteins after they have been synthesized

DNA DAMAGE MECHANISMS

Endogenous Agents

• Spontaneous chemical reactions

1. Deamination: nucleotides lose amine groups
   • Cytosine –> uracil
   • Adenine –> hypoxanthine
2. Depurination: purine (adenine or guanine) released from DNA
   • Bond between deoxyribose and purine base spontaneously cleaves
   • Produces AP site (apurinic site)

Exogenous Agents

• Exposure to mutagens (chemicals or radiation)

1. Pyrimidine dimers: induced by UV light exposure
   • Cyclobutane ring forms between adjacent pyrimidines (often thymines)
   • Distorts the DNA double helix
2. Alkylation: addition of methyl/ethyl groups to nucleotides
   • -CH3 or –CH2CH3 add to nitrogenous bases at numerous positions
3. Bulky group addition: exposure to carcinogens
   • i.e. benzo(a)pyrene: aromatic, polycyclic structure can react with purines/pyrimidines at numerous positions
   • Cause distortions in DNA helix

Carcinogen

• Cancer-causing mutagen

CONSEQUENCES OF DNA DAMAGE
• Can increase frequency of mutations
• Mutations: nucleotide substitutions, deletions and insertions

CLINICAL CORRELATIONS

Skin melanomas

• Pyrimidine dimers produce helical distortions that result in skin cancers

Cigarette smoking

• Carcinogens in smoke form covalent bonds with DNA
• Disrupts H-bonding between nucleotides: causes frameshift
• Frameshift changes subsequent codons in DNA strand
• Constant exposure to carcinogens –> lung cancer

REPAIR MECHANISMS
• Mismatch-repair: fixes replication errors missed by DNA Polproofreading (cannot repair damage)
• Base excision repair: deamination, depurination and alkylation
• Nucleotide excision repair: pyrimidine dimers and bulky group addition