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BIOL 308
Dragana Miskovic

1. What is the role of Shine-Dalgarno sequence? It’s a conserved sequence of nucleotides (AGGAGGU) 8-13 nt upstream from the first codom to be translated. It functions as a ribosomal binding site and binds the mRNA to the ribosome (pairs with the sequence at the 3’ end of the 16S rRNA of the small 30S subunit). It also aligns the ribosome at the correct position for initiation of translation. 2. What is the role of Kozak sequence? It’s a sequence that occurs on Eukaryotic mRNA (CCRCCAUGG; R=A or G). The sequence is recognized by the 40S ribosomal subunit (with tRNA already charged) of Eukaryotes. It helps differentiate start codons from other methionine (AUG) codons. (It’s a eukaryotic Shine-Dalgarno sequence) 3. What are the major differences in initiation of translation between eukaryotes and prokaryotes (remember: first AA and the way mRNA and tRNA bind to ribosomes)? **************** 1. In Eukaryotes, the tRNA cariies a regular methionine which is added like any other amino acid, and then excised later if it’s not part of the code. On the other hand, Prokaryotes add a formyl group (CHO) to the methionine and therefore, carry fMet (formyl methionine) which is easily recognizable from other methionines. 2. The 40S ribosomal subunit of ribosomes recognizes the Kozak sequence in Eukaryotes. The methyl cap interaction also plays a role in the binding. However, in prokaryotes, the Shine-Dalgarno sequence binds to the 30S ribosomal subunit. In eukary, tRNA forms ternary complex before binding to ribosome (in pro, just tRNA binds to P site). In euk, 43S binds to mRNA through 5’ methyl cap and Kozak sequence (in pro, 16S interacts with Shine-Dalgarno). In euk, 3 factros steop binding of 60S subunit => 3,4C and 1A (in pro, only IF3 prevents binding). N.B: the similarity is that both Eukaryotes and Prokaryotes have 2 types of methionine tRNAs charged with the same enzyme – methyonil tRNA synthetase. One functions to deliver internal methionines and the other to deliver the ones required for initiation. 4. Describe in detail events during initiation of the Eukaryotic (or Prokaryotic) translation. ***** Prokaryotic: • IF3 binds to free 30S subunit (prevention of binding of 50S subunit) • IF1 binds (prevents potential binding of tRNA to A site) • IF2 (GTPase) complexes with GTP and binds • mRNA binds to 30S subunit through interaction of Shine-Dalgarno sequence with 16S rRNA • Initiator tRNA binds (anticodon-codon base pairing) to P site • IF3 is not needed any more and is released This is 30S initiation complex • 50S subunit binds • This displaces IF1 and IF2, and GTP is hydrolyzed –energy consuming step This is 70S initiation complex and translation can start. N.B: polycistronic mRNAs have multiple internal Shine-Dalgarno sequences resulting in synthesis of different proteins simultaneously Eukaryotic: Preinitiation complex forms as 40S, eIF1,3,4 combines  mRNA binds using 5’ cap  eIF1,3,4 released when AUG is found through Kozak sequence  eIF5 displaces 2,3 and brings in GTP for E  eIF4 helps ribosomes combine  eIF2-GDP is recycled by eIF2B to control the rate of translation 5. Briefly explain two cases of RNA having enzymatic capability (how many ribozymes did we mention in class)? 1. Peptidyl transferrase: Responsible for the formation of peptide bonds and therefore, elongation of the polypeptide chain. 2. 23S rRNA of the large ribosomal subunit and since they are part of ribosomes, ribosomes are also considered as ribozymes. 3. Self splicing introns of Group I and II introns. 4. snRNAs involved in splicing. (U1, U2, U3, U4, U6) 5. RNAse P cleaves primers 6. What is a peptidyl transferase? What is catalyzed by peptidyl transferase? It’s an aminoacyltransferase. It is responsible for the main function of ribosomes i.e. formation of peptide bonds during the elongation stage of translation. It connects AA on ribosomes and dissociates AA from tRNA during translation. The activity is carried out by an RNA and is therefore a ribozyme. 7. How is gene expression controlled at the level of translation (mRNA is now in contact with ribosomes or their subunits/translation factors; three things mentioned in the class)? *** (Dra’s answer: Gene is controlled through phosphorylation of translation factors, multiple AUG codons and IRES (internal ribosomal entry site). Phosphorylation slows down translation and it occurs as lack of AA leads to slowing down of eIFB – eIF2-GDP recycling. Multiple AUG through leaky scanning of weak AUG Kozak sequences or AUG-UGA combinations that slow down translation. Leaky scanning produces protein without signal sequence that helps with leading the molecule to its destination. AUG-UGA combination produces non-functional. IRES is mRNA without 5’ cap and helps with controlling balance between cell death and birth.) 1. Phosphorylation of translation factors: such as that of eIF2 that down regulates translation initiation: a. Amino acid starvation – accumulation of uncharged tRNAs b. Abundance of uncharged tRNAs activates protein kinase responsible for phosphorylation of eIF2 c. This protein kinase phosphorylates eIF2 d. Phosphorylated eIF2 cannot be recycled by eIF2B Outcome: when amino acid level is low translation is going to slow (or shut) down 2. Multiple AUG codons: a. AUG/UGA combo: they are short ORFs between 5’ end of the mRNA and the beginning of the actual gene aka upstream ORF or uORF. The ribosome binds to them and translates them but then drops off at the stop codon and therefore, the actual gene’s translation is down regulated. b. Weak Kozak sequence: If the kozak sequence is weak, the translation machinery will be binding to many AUG sites and translating faulty proteins. This leads to a down regulation of the actual gene. 3. Internal Ribosome Entry Sites (IRES): they are nucleotide sequences that allow cap-independent translation i.e. they allow for translation initiation in the middle of the mRNA sequence thus controlling the level of translation. 8. Which two factors greatly influence efficiency of protein synthesis? 1. Simultaneous translation of a single mRNA by multiple ribosomes. 2. Circular mRNA (from Dra.. No idea what it means) 3. Rapid recycling of ribosomal subunits.
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