Bio 230 exam 2 weeks 4 and 5

involves cleavage of the new transcript followed by template-independent addition of adenines at its new 3' end, in a process called polyadenylation

• 5’ cap: added to the 5’ end of an immature mRNA strand during transcription for nuclear export and to protect the transcript (mRNA) from being broken down (mRNA degradation)
• Spliceosome removes introns
• 3’ poly A tail: added to the 3’ end of an immature mRNA to help protect mRNA from degradation and promote nuclear export

• Spliceosome is a complex made up of snRNPs
• snRNPs contain protein and RNA
• RNA: bind to conserved regions of introns
• Protein: cut introns
• Interactions between snRNPs and additional proteins causes the intron  to “bend”
• Conformational changes to the protein causes more bending and facilitates intron splicing via a chemical reaction

• multiple proteins synthesized from one mRNA by varying the exon composition of that same mRNA.
• Additional regulatory proteins bind to conserved regions and “mask” them from snRNP recognition
• Results in “exon skipping”
• Alternative splicing occurs from exons being skipped, exons being extended, or introns being retained
• Alternative splicing increases complexity of gene expression

• Mature mRNAs transported out of the nucleus
• Ribosomes translate mature mRNAs either in cytoplasm or in ER

• Cytoplasm: if a protein is made in the cytoplasm it stays in the cytoplasm; inner peripheral proteins 
• ER: proteins only made in the ER are made for export; integral-membrane proteins, outer-peripheral proteins 
• Anterograde transport: from ER to plasma membrane via a vesicle 
• Retrograde Transport: from plasma membrane to ER via the golgi (EX=ricin)

• Ribosome: ribonucleoprotein that is composed of rRNA and proteins AND has ribozyme activity
• Ribozyme is an enzyme made of RNA that can catalyze a reaction
• Large subunit (60S): binds tRNA
• Small subunit (40S): binds mRNA
• Ribosome has 3 sites for tRNA to bind: E P A, which are oriented 5’ 3’ bc ribosomes move towards 3’ end of mRNA
• aminoacyl site (abbreviated A), the peptidyl site (abbreviated P) and the ex

• tRNA: single strand of RNA that has secondary structure mediated by hydrogen bonds
• contains: anticodon region and amino acid binding region (acceptor stem) 
• the anticodon region binds to the mRNA codons to synthesize the protein and the acceptor stem binds the AA
• tRNA carries AA to make the polypeptide chain
• tRNA can be “charged” or “uncharged.” Charged tRNA’s have the AA bound to its acceptor site (and can thus participate in translation) while uncharged tRNAs do not have an amino acid bound, charged tRNA completed by aminoacyl tRNA synthase

• multiple codons for one amino acidthere are more codons to recognize than there are tRNAs to recognize them
• This redundancy is often seen in the 3rd position of the tRNA and this position is called the “wobble base pair” for that reason. This affects pairing because many codons can still code for the same amino acid. This is helpful for the cell because if there is a mutation in the 3rd position, the cell can still conserve the same amino acid during protein translation. For the cell to get this redundancy there are 2 main mechanisms: 1) there is less steric hindrance at the 3rd position which can allow non-canonical binding 2) anticodons have the base inosine (I) which binds to U, C, and A

• G-proteins: guanine nucleotide-binding proteins — act as molecular switches inside cells: cause other molecular events to occur
• Initiation, elongation and release factors are examples
• G-proteins bind to GTP = ”active” conformation
• G-proteins hydrolyze GTP = ”inactive” conformation

• GTPase activating protein
• inactivates G-proteins by binding to activated G proteins and stimulate their GTPase activity, with the result of terminating the signaling by hydrolyzing GTP to GDP

• pre-mRNA to mature RNA takes place in nucleus
• Small ribosomal units bond to the 5' cap of mRNA with the help of initiation factors and tRNA-Methionine. Then the complex moves to the first AUG codon. When the Methionine-tRNA finds the codon, initiation factors hydrolyze GTP to provide energy to form the ribosome. Finally, the initiation factors are released and the Large subunit binds

Elongation factor-1 transports aminoacylated tRNA to the A site. Then GTP hydrolyzes with the correct pairing because of the stable bonds. Then Elongation-Factor 2 moves tRNA from the P site to the E site. This occurs after the peptide bond forms and the A site becomes vacant for the next tRNA to translate

Termination occurs when a stop codon is encountered (UGA, UAA, or UAG). A release factor (not tRNA) binds to the codon at the A site. This triggers hydrolysis of GTP and of the new peptide chain. After hydrolysis, the ribosome complex dissociates

• Primary: Amino acid sequence
• Secondary: Hydrogen bonds of backbone
• Tertiary: Interactions between R-groups
• Quaternary: Multiple subunits

• Certain amino acids tend to form helices
• Most prevalent secondary structure
• Backbone that interact via hydrogen bonds to form helix

• Certain amino acids tend to form sheets or strands
• Secondary structure arises via hydrogen bonding along backbone

• Supersecondary structure occurs when 2-4 secondary structures interact, results in structural motifs
• Mediated by hydrogen bonds, disulfide bonds (covalent) and/or hydrophobic interactions
• Supersecondary structures contributes to tertiary structure
• Structural motifs cannot function without rest of protein

• Protein domains:
• Domains dictate the functionality of a protein
• Proteins can have one, many or even no domains (rare)
• Can function without rest of protein

• ricin glycosidase: Remove a purine from RNA
• Quaternary structure
• A-chain of ricin: inactivates ribosomes, inhibiting protein synthesis by blocking the binding of elongation factors.
• B-chain of ricin: binds to carbohydrates
• If a protein is misfolded during translation and cannot be fixed, where is it transported? It is transported to the cytoplasm for destruction via the Seq61 protein, Seq61 allows ER-cytosol transport

• 2 fatty acid chains (uncharged, hydrophobic), glycerol, phosphate group (negatively charged, hydrophilic)
• Amphipathic, Saturation, Double bond

This means that they have a hydrophilic, polar phosphate head and two hydrophobic fatty acid tails. These components of the phospholipids cause them to orientate themselves, so the phosphate head can interact with water and the fatty acid tails can't, hence forming a bilayer

Phospholipids in the lipid bilayer can either move rotationally (with help of an enzyme), laterally in one bilayer, or undergo transverse movement between bilayers. Lateral movement is what provides the membrane with a fluid structure

• Fluidity can be regulated by “lipid packing”
• Packing = how closely the fatty acid chains can interact in the layers
• Based on length and saturation
• More fluid = less interactions

• Higher lipid length decreases fluidity
• Unsaturated double bonds = more fluidity bc kinks are created

• Fluidity is determined by membrane’s melting temperature
• Heat increases = membrane changes from rigid state to more fluid state
• Low melting temperature = presence of fatty acids that promote fluidity
• High melting temperature = fatty acids and cholesterol that restrict fluidity
• Less rigid allows for more movement, which means more fluidity
• Saturated fatty acids = higher melting temperature bc residues will react with each other causing the fatty acid to be in more rigid state thus less fluidity

• Cholesterol is amphipathic
• Cholesterol acts as a regulator of membrane fluidity
• High temps = raises MT (less fluidity)
• Low temps = prevents phospholipids from clustering together (interacting) (more fluidity)

Permanent membrane residents

Temporary membrane residents

• Anchored to membrane via lipidated (acyl) amino acids
• Can be permanent or temporary

• involves the addition of a “lipid anchor” to the protein to help associate the protein with the membrane
• Anchors the protein strongly to the membrane

• Channel-linked receptor
• Enzyme-linked receptor
• G-protein coupled-receptors
• Intracellular receptors

• Channel-linked receptor
• Binding opens a protein channel

• Enzyme-linked receptor
• Binding activates an enzyme
• Kinase, phospholipase, phosphatase are common

• G-protein coupled-receptors
• Binding activates a G-protein

• Intracellular receptors
• Activation causes intracellular changes

• Translation into ER produces proteins for exocytosis (secretion), integration into membrane or other membranes of the endomembrane system
• translated in the ER after translation starts in cytoplasm
• After transport to translocon, translation will continue into the ER until a hydrophobic stop sequence is translated.
• Remaining protein will be translated in cytosol
• Fusion of the vesicle with the plasma membrane results in the protein being positioned in the plasma membrane.
• Some peripheral proteins undergo post-translation modifications (via enzymes in the ER) during transport. Lipidation is an example