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Updated SQ lec 28 - 35.docx

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University of Waterloo
BIOL 308
Dragana Miskovic

STUDY QUESTIONS: LEC 28-35 1. List and briefly explain different mechanisms of post-transcriptional control of gene expression (think about examples)? - Localization of mRNA  positioning of mRNA close to the sites where the protein produced from mRNA is required. Efficient way to unevenly distribute proteins in polarized cell. E.g., maternally inherited mRNAs such as bicoid are asymmetrically distributed in embryo. - RNA editing  RNA alteration in mRNA sequences. E.g., in mitochondria of protozoa, and mitochondria and chloroplasts in plants, there is addition or deletion of U residues. - Post-transcriptional silencing by siRNA or miRNA  RNA intereference – ability of small ds or ss RNA molecules to induce sequence specific gene silencing. miRNAs work as regulatory factors in development e.g., in C elegans, larval protein (LIN14) is detected only earl in development. - Translational control switch coordinate and opposite translational regulation of ferretin and transferrin receptor. mRNAs for both proteins have IRE which binds iron-responsive binding protein IRP which is sensitive to iron concentration. - RNA stability (degradation and stabilization)  balance between mRNA degradation and synthesis determines the level of individual mRNAs in cells. 2. Explain the role of mRNA stability (or editing, or iRNA, or translational control switch or mRNA localization – five different questions possible) in control of gene expression. mRNA stability is important because translation will only occur if the mRNA is not degraded and if the ribosome can reach the mRNA and begin translation. With the translational control switch, the presence of a certain nutrient can affect whether the mRNA is degraded or not (transferrin) or translation is inhibited (ferritin). iRNA can be made to inhibit mRNA translation by binding regions through a miRNP or siRNP complex. 3. Explain the connection between pre-mRNA splicing and transport of mRNA from the nucleus. There is some kind of mechanism that normally prevents pre-mRNA from exiting the nucleus before it is spliced. This was illustrated in an experiment where hsp30 mRNA was not observed unless cells were treated with cyclohexamide. This caused protein synthesis inhibition, resulting in hsp30 mRNA being visible due to the mechanism for preventing pre-mRNA exiting the nucleus being inhibited. 4. What is trans-splicing? Give an example. Trans-splicing is the creation of multiple monocistronic mRNA from a single polycistronic pre- mRNA. An example of this is in the Trypanosoma, where the leader sequences are transcribed, capped, and spliced to form mini exons; these are then trans-spliced to polycistronic transcription units which are spliced, after which a poly-A tail is added to the form the final product. 5. What does S in 16S stand for? What is the numerical value of this constant? -13 S stands for svedberg units, and 1S = 10 s. It is a measure of sedimentation rate of suspended particles when centrifuged under constant conditions. It depends on both size and shape of the particle. It is a good measure of relative size if one is comparing same types of molecules. 6. What are the roles of three major RNAs in protein synthesis? mNRA – carries genetic information from DNA in the form of codons. tRNA – translates mRNA code. Each AA has its own tRNA. Binds to mRNA due to codon- anticodon complementarity. rRNA – associates with proteins to form ribosomes which catalyze the assembly of protein chains. 7. What is the name of the region of tRNA molecule which attaches to an amino acid? CCA sequence of acceptor stem. 8. How many different tRNAs are there in an eukaryotic cell? How many different aminoacyl tRNA synthetasis are there in an eukaryotic cell? 30-40 different tRNAs in bacteria; around 50 in animals and plants. There are 20 aminoacyl-tRNA synthetase that recognize tRNA – one enzyme for each of the 20 essential AA. 9. What are the roles of tRNA in translation? Bind appropriate amino acids and bring them to appropriate sites matching mRNA codon during translation. 10. What is the wobble position for an anticodon? For a codon? Position 3 on the codon and position 1 on the anticodon. Some tRNAs can have a nucleotide in this sequence that does not have to properly base pair for the amino acid to be transferred, as there are 61 possible codons for amino acids and only 50 tRNAs. 11. Explain what does it mean when we say that the code is degenerate? Codons are synonymous? The code is degenerate as in there are possibly several codons coding for a single amino acid, and so there may be a large number of nucleotide sequence possibilities that code for the same amino acid sequence. Codons are synonymous in that there is more than one codon coding for the same amino acid in many cases. 12. How are ribosomal subunits held together? What could be used to separate subunits? Ribosomal subunits are held together by divalent cations, Mg. They can be separated through EDTA as well as high salt concentration. 13. What is the role of Shine-Dalgarno sequence? (EXAM) 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. 14. 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) 15. 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)? - In Eukaryotes, the tRNA cairies 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. - 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 factors stop binding of 60S subunit => 3,4C and 1A (in pro, only IF3 prevents binding). - In eukaryotes, the tRNA binds BEFORE the preinitiation complex binds the mRNA. In prokaryotes, the preinitiation complex binds mRNA, and then initiator tRNA enters the P site. 16. 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 result
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