SEARCH
You are in browse mode. You must login to use MEMORY

   Log in to start

level: Level 1

Questions and Answers List

level questions: Level 1

QuestionAnswer
Other cytosolic post-translational modifications1. Proteolytic cleavage, e.g. to activate a protein. 2. Addition of lipids to permit membrane targeting. 3. Phosphorylation. 4. ADP ribosylation. 5. Methylation
Mechanistic features of translationInitiation Elongation – including translocation Termination
Prokaryote mRNA (E. coli)The start signal is AUG (or GUG) preceded by several bases that pair with 16S rRNA. The purine-rich region, called the Shine-Dalgarno sequence is complementary to the initiator sites of mRNA
Methionine accepting tRNAs in E. coliVontains two types of tRNAmet. tRNAfmet and tRNAmmet. tRNAfmet : Met residues attached to this are formylated, Initiate polypeptide chains only, Recognises AUG and GUG. tRNAmmet : Met residues are only attached, not formylated, Recognises the codon AUG only, Used as a source of internal met residues
Translation Initiation in Prokaryotes: 30S initiation complexIF-3: 22 kDa. Binds to the 30S subunit and prevents association with the 50S subunit. IF-1: 9 kDa. Binds near the A site therefore directs fmet-tRNA to the P site. IF-2: 120 kDa. Reacts with fmet-tRNA and GTP to form a ternary complex IF-2-GTP-fmet-tRNA. Delivers the ternary complex and mRNA to the partial P site in the 30S subunit-mRNA complex. Triggers GTP hydrolysis when the 50S subunit joins the complex. It does not recognize met-tRNA or any aa tRNA used for elongation.
Translation Initiation in Prokaryotes: 70S initiation complexThe 50S subunit joins the 30S initiation complex to form the 70S initiation complex. Initiation factors are released and GTP is hydrolysed.
Elongation: OverviewThree steps: 1.Codon-directed binding of the incoming aminoacyl-tRNA. 2. Peptide bond formation 3. Translocation (movement) of the ribosome along the mRNA in a 5’ -> 3’ direction by the length of one codon
Peptide bond formation by peptidyl transferaseThe aminoacyl portion of fMet-tRNA is transferred to the amino acid group of the aa residue in the A site, forming a peptide bond. Activity due to the ribozyme function of 23 S. Overall Keq ~1. therefore no energy input is required
Mechanism of translocation in elongationElongation Factors: EF-Tu, brinds in each AA-tRNA. EF-G, facilitates translocation to the next codon.
EF-TBinding of the incoming aminoacyl-tRNA requires a soluble supernatant factor, elongation factor T (EF-T) and GTP. EF-T is composed of two polypeptides: EF-Tu (heat unsteable) and EF-Ts (heat stable) EF-Tu is very abundant. Most aa-tRNAs in the cell are complexed with EF-Tu. EF-Tu does not react with fmet-tRNAmet explaining why this is not bound during elongation. When bound to EF-Tu the labile ester bond between the tRNA and aminoacyl residue is protected from hydrolysis.
Proofreading role of EF-Tu in translationIt takes a few milliseconds for GTP hydrolysis to occur, and a few more milliseconds for EF-Tu-GDP release. Only after EF-TU-GDP release can peptide bond formation occur. These intervals provide the opportunity for a weakly bound, non-cognate aa-tRNA to dissociate from the ribosome.
Mechanism of translocationEF-G/GTP binds to the pre-translocation ribosome at a site including L7/L12, L11 and the sarcin/ricin loop of 23S rRNA. The tRNA-like domain interacts with the 30S subunit close to the partial A site. GTP hydrolysis induces a conformational change in EF-G, forcing its arm deeper into the 30S subunit, which forces the peptidyl tRNA from the A site into the P site, carrying the mRNA and deacylated tRNA with it. RESULT:-ribosome moves along the mRNA by length of one codon
Elongation stepsEF-Tu-GTP places the aminoacyl-tRNA on the ribosome and then is released as EF-Tu-GDP. EF-Ts is required to mediate the replacement of GDP by GTP. The reaction consumes GTP and releases GDP. The only aminoacyl-tRNA that cannot be recognised by EF-Tu-GTP is fMet-tRNAf, preventing use internally
Termination1. Release factors: RF1 UAA + UGA, RF2 UAA + UAG, RF3 –GTP aids binding. 2. RF binds vacant A site. 3. Peptidyl transfer of the peptidyl group to water, rather than an aminoacyl tRNA. 4. Hydrolysis of RF3- GTP to GDP dissociates everything.
Initiation of protein synthesis in eukaryotesAUG is almost always used as the initiation codon. A special initiator tRNA, tRNAimet is used as the initiator. (Does not become formylated). tRNAmmet is used to insert internal methionines. The “first” AUG is usually use for initiation (~90%). Context dependent.
The cap binding complex (eIF-4F) in eukaryoteseIF4E binds 5’ cap. eIF-4A is an ATP-dependent RNA helicase that removes secondary structure near the 5’ end. Needed for scanning movement of the 40S subunit along the mRNA. eIF-4G is a 'scaffold' subunit. links together the initiation complex. Cleavage by protease results in inhibition of initiation.
Initiation of protein synthesis in eukaryotesUnlike prokaryotes, the Met-tRNAi is pre-bound to the 40S subunit, i.e. it’s binding is not codon-directed. The mRNA is SCANNED to find the first AUG. The ATP requirement is for the helicase activity of eIF-4A to remove hairpin structures in the mRNA. Polysomes in electron micrographs are often observed to be circular. Poly (A) tail greatly stimulates rate of protein synthesis. Poly (A) tail binds poly (A) binding protein (PAB1). PAB1 interacts with eIF4G and eIF4E bound to “cap”. Ribosomes that have completed translation dissociate into subunits. These can readily find the nearby m7G cap and initiate another round of protein synthesis. Rapid recycling of subunits increases the efficiency of translation.
Internal Ribosome Entry Sites (IRES)The vast majority of eukaryotic mRNAs are translated through the ribosome scanning mechanism. An alternative mechanism is internal ribosome entry. The mRNAs lack a 5’ cap, and translation is initiated at internal ribosome entry sites (IRES). IRESs have a complicated tertiary structure and bind 40S subunits in close proximity to an AUG codon.
Regulation of protein synthesis during the cell cycleG2/M transition decreases 75% total protein synthesis. Caused by cell cycle-dependent dephosphorylation of eIF-4E (Cap binding). Decrease affinity of ribosomes for the cap. IRES-containing RNAs are unaffected. Relative IRES translation rates increase in M phase. In apoptosis, eIF-4G is cleaved: caspase 3, all translation decreases.
PicornavirusesShuts off ~ 90% of host protein synthesis. Gives virus maximum competition with the host. Viruses use IRES, so cell cycle independent.
Release of polypeptide chainsThe water molecule bound to the release factor hydrolyses the ester bond in the peptidyl tRNA, releasing the completed polypeptide. NB. During normal chain elongation , water is excluded from the peptidyl transferase centre of the ribosome.
Translational control mechanismsRegulation of the activities of initiation and/or elongation factors by phosphorylation (pro- and eukaryotes). Blocking / opening of ribosome binding sites by reversible changes in secondary structure (prokaryotes). Autogenous regulation. Protein product of a gene binds to ribosome binding site in mRNA, preventing initiation (prokaryotes). Reversible binding of a repressor protein to a response element in 5’ UTR (eukaryotes). Differential stability of mRNA.
Autogenous control of ribosomal protein synthesis in E. coliThe demand for r-proteins is growth rate dependent (high demand during rapid growth, low demand during slow growth). It is also closely coupled. R-protein synthesis is regulated at the level of translation. Which is shown by transforming E. coli with a high copy plasmid encoding r-protein. ─ mRNA level increases greatly, but not the amount of r-protein. Ribosomal protein genes are organised into several operons, each containing up to 11 genes for r-proteins. Some of these operons also encode non-ribosomal proteins whose synthesis is growth rate dependent.
Regulation of translation of mammalian ferritin and transferrin mRNAs by iron-response element binding proteins.Ferritin is a cytosolic protein that binds iron ions and prevents accumulation of toxic levels of Fe2+/Fe3+. However when Fe is limiting, ferritin poses a problem – it competes for Fe with iron-requiring enzymes. Mammalian cells therefore modulate the synthesis of ferritin – expressed under excess Fe, repressed under Fe scarcity. The transferrin receptor (a cell surface protein responsible for Fe uptake into cells) shows reciprocal regulation of synthesis to that of ferritin.
Eukaryote mRNA decayNearly all mRNAs subjected to poly (A) tail shortening. When tail < 30 A’s residues in length, Poly (A) binding protein is lost and 3’ end no longer associates with cap. This leads to decapping followed by degradation. Cleavage at the endonuclease cleavage site in the 3’ UTR results in decapping followed by degradation.
Starting point: in a eukaryotic cell, most protein synthesis occurs in the cytosol1.A common pool of ribosomal subunits in the cytosol. 2.Is used to assemble ribosomes on mRNAs encoding cytosolic proteins: these remain free in the cytosol. 3.Multiple ribosomes assemble, producing free cytosolic polyribosomes (polysomes). 4.Newly made proteins are released to the cytosol once their synthesis is complete and the ribosomes are dismantled, returning back
Nascent proteins are crowdedSince a single mRNA is translated at the same time by multiple ribosomes, nascent proteins are extruded in close proximity. They are therefore in danger of aggregation. Nascent proteins are in a non-native, aggregation-prone conformation
Molecular chaperones are needed to favour correct folding, not aggregation.1. Hydrophobic patches on nascent/unfolded proteins are recognised by Heat shock protein 40 family members, which… 2. …deliver the substrate to ATP-bound (OPEN conformation) Heat shock cognate protein 70 (Hsc70 chaperone) and stimulate the ATPase activity of Hsc70… 3. …resulting in ADP-bound (CLOSED conformation) Hsc70 shielding the hydrophobic patches of the substrate, preventing aggregation, and allowing time for the hydrophilic parts of the substrate to fold. 4. Upon nucleotide exchange, Hsc70 adopts its open conformation, releasing the substrate, with folded soluble structures: this partly folded protein may now snap into its final conformation.
How do we study chaperone interactions?1. Heat target protein (PrT) at 45C for 15 min and separate aggregated (P, pellet) and soluble (S) fractions by centrifugation. SDS-PAGE, silver stain and quantify. Most is insoluble. 2.Heating in the presence of Hsp40 increases the S fraction. 3. Heating in the presence of Hsc70 has a larger effect 4. Hsp40 and Hsc70 together have even more effect 5. Maximal solubilisation requires Hsp40, Hsc70 and ATP
Hsc70 actionHsc70 does not actively FOLD a protein, but binds and shields its hydrophobic regions and thus prevents AGGREGATION of nascent or unfolded proteins. A partially-folded Hsc70 client protein may be: released, and find its stable conformation, passed on to other chaperones for further folding, released, in preparation for transport, or passed to proteasomes for degradation
How are protein clients released from Hsc70?A nucleotide exchange factor (NEFs eg. BAG-1,2) binds the Hsc70: client complex and removes ADP from the nucleotide-binding site of Hsc70. This promotes NUCLEOTIDE EXCHANGE, allowing entry of ATP into the nucleotide binding site of Hsc70. Hsc70:ATP adopts an OPEN conformation, releasing the client protein.
Hsc70 co-chaperones can pass partially-folded substrates to other chaperones and chaperonins for further folding or multimeric assembly.Hsp90 can provide a platform for further protein folding and also assembly of multimeric complexes. Chaperonins provide a cage that isolates small (<70 kDa) folding proteins (e.g.tubulin, actin) from the cytosol. Residence time ~10s.
The FATE of chaperone client proteins is not pre-determinedThe prevailing concentrations of Hsc70 and Hsp90 co-chaperones determine the proportion of an unfolded/misfolded protein that can gain a stable conformation and the proportion that is destroyed. n other words: co-chaperones make decisions by competing to release chaperone clients. There is an interacting, competing network of co-chaperones that determines the fate of a chaperone client
Architecture of the proteasomeProteasomes are abundant in the cytosol and nucleoplasm. The 20S core particles are cylinders with three proteolytic activities: chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolysing (caspase-like). The active sites are inside the barrel, encoded by the β subunits. Each 20S core has 19S caps, regulatory particles (RP) at one or both ends.
How is a protein targeted to the proteasome?Ubiquitin (Ub) is a conserved 76 amino acid protein found in all eukaryotic cells. Cytosolic proteins destined for proteasomal degradation are usually marked for destruction by covalent addition of a chain of Ub molecules (polyubiquitylation) allowing them to be bound by the 19S RP. A chain of four Ub proteins means a doomed protein: tetra-Ub is a degradation signal.
Ub Mechanism1.Ub is activated by an E1ubiquitin-activating enzyme. 2.Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme. 3.Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme. 4.Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme. 5. and transfers Ub to the target
How is the protein targeted to the proteasome? (pt.2)Ubiquitin is normally added covalently to the side chain of an available LYSINE residue on the target molecule. The process is repeated using side chains of lysine residues in ubiquitin, until a chain of at least 4 Ub is completed. This multi-ubiquitin tag is a degradation signal. Polyubiquitylated proteins can be bound by the proteasomal 19S RP.
Targeting the proteasome and destruction1. Polyubiquitylated proteins bind the 19S regulatory particle of the proteasome 2. The RP uses ATP to generate energy to unfold the target protein and feed it into the 20S core. Deubiquitylases (DUBs) remove Ub molecules and return them to a common pool for recyclin. 3. Three proteolytic activities are encoded by the β subunits of the 20S core 4. The target protein is degraded into small peptides, which are ejected from the proteasome.
And when components of the UPS fail?Proteins that would normally be destroyed accumulate instead. This can lead to the formation of aggregates e.g. in neurons of people with Parkinson’s and Alzheimer’s. If cell cycle proteins are not degraded properly, it can lead to cell proliferation (as in cancer).
Other cytosolic post-translational modifications1. Proteolytic cleavage, e.g. to activate a protein. 2. Addition of lipids to permit membrane targeting. 3. Phosphorylation. 4. ADP ribosylation. 5. Methylation