Assignment 12-key PDF

Preview

Summary

Assignment 12 key...

Description

Assignment 12

MBB 222 Summer 2020 (Craig)

Answer the questions in your own words and to the best of your ability. Upload as WORD or PDF files to Canvas by Mon. Aug. 3, 2020, 9:00 am. This assignment will be graded for completion only. Max. 1% of final grade. 1. The first part of a gene sequence encoding Protein X is shown below, starting at the +1 nucleotide. Show the mRNA sequence. Use the Genetic Code table to show the protein sequence for this gene segment. What would you expect to happen to the protein sequence if the underlined C in this gene were mutated to an A? What if the underlined A was changed to a T? What if an insertion of a G occurred immediately after the underlined A? Note: the Genetic Code table will be provided for Exam 4. 5’-ATG TTA TCC GAG GCC CCT ATT AAG ATG ACC GAC TAT AGC … -3’ mRNA 5’-AUG UUA UCC GAG GCC CCU AUU AAG AUG ACC GAC UAU AGC … -3’ protein N-Met-Leu-Ser-Glu-Ala-Pro-Ile-Lys-Met-Thr-Asp-Tyr-Ser -… A C→A mutation would change the GCC codon, which encodes for Ala, to a GCA codon, which also encodes for Ala. This is known as a “silent mutation” because it does not affect the protein sequence. An A→T mutation would change the UAU codon, which encodes for Tyr, to a UUU codon, which encodes for Phe. This is a “conservative” amino acid change, since both residues are bulky hydrophobic aromatic amino acids. It will likely have no effect on the protein structure or function. An insertion of a G after A would result in a frameshift, giving the following DNA sequence: 5’-ATG TTA TCC GAG GCC CCT ATT AAG ATG ACC GAC TAG TAG C … -3’ The mRNA sequence will be as follows: 5’-AUG UUA UCC GAG GCC CCU AUU AAG AUG ACC GAC UAG UAG C… -3’ The second to last codon, UAU, which encodes Tyr, will become the stop codon UAG, which will signal termination of protein synthesis, to produce only a short peptide rather than an intact protein. Typically this truncated protein will not fold correctly, will aggregate and get degraded by cellular proteases. Note, the insertion actually introduces 2 new stop codons, UAG, UAG.) protein N-Met-Leu-Ser-Glu-Ala-Pro-Ile-Lys-Met-Thr-Asp-C 2. Define the following terms (3): (a) anticodon (b) tRNA synthetase (c) stop codon (a) anticodon – the triplet sequence in the anticodon loop of tRNA that base pairs with the codon in mRNA (b) tRNA synthetase – an enzyme that catalyzes the covalent attachment (coupling) of an amino acid to the 3’ end of its cognate tRNA. (there is a tRNA synthetase for every amino acid) (c) stop codon - signals the end of protein synthesis (does not specify an amino acid, does not have a corresponding tRNA with a complementary anticodon) 1

Assignment 12

MBB 222 Summer 2020 (Craig)

3. List the start codon, all the stop codons, and all codons that specify the amino acids Pro, Met, Ser, Trp and Tyr. Write out the anticodon sequence (5’ to 3’) for tRNAs that bind Trp (tRNATrp) and Met (tRNAMet). Start - AUG Stop - UAA, UAG, UGA Pro - CCU, CCC, CCA, CCG Met – AUG (same as the start codon) Ser - UCU, UCC, UCA, UCG, AGU, AGC Trp - UGG Tyr - UAU, UAC tRNATrp anticodon: 5’ CCA tRNATyr anticodons: 5’ CAU 4. Provide numbers for the following in a typical eukaryotic cell. Explain your answer where necessary/appropriate. (a) Number of naturally occuring amino acids? 20 (b) Number of tRNA synthetases? 20, one for each amino acid. Each tRNA synthetase binds only one amino acid but some can recognize more than one tRNA, all of which are specific for the same amino acid but which have different anticodons and thus recognize different codons (see (c)). (c) Number of serine codons? Six: UCU, UCC, UCA, UCG, AGU, AGC. (d) Number of tRNAs that can bind to the UAG codon? None – this is a stop codon. Release factors (proteins) probably bind to this codon. (e) Number of different amino acids that can be inserted at the UCG codon? One – Ser. 5. List the names of the small and large subunits for both prokaryotic and eukaryotic ribosomes. What component of bacterial ribosomes recognizes the Shine-Dalgarno sequence? Which component of bacterial ribosomes catalyzes peptide bond formation? Prokaryotes

Eukaryotes

Small subunit

30S (16S rRNA, 21 proteins)

40S (18S rRNA, 33 proteins)

Large subunit

50S (23S rRNA, 5S rRNA, 34 proteins)

60S (28S rRNA, 5.8S rRNA, 5S rRNA, 49 proteins)

70S

80S

Complete ribosome

The 16S rRNA of the small (30S) subunit of bacterial ribosomes binds the Shine-Dalgarno sequence. The 23S rRNA of the large (50S) subunit catalyzes peptide bond transfer. 6. Describe the functions of each of the following components in bacterial protein synthesis: (a) tRNA – transfer RNA - the adapter molecules that translate the mRNA sequence into a protein sequence; has an L-shaped tertiary structure; tRNA combines specifically with an amino acid and positions the amino acid in the ribosome such that it will form a peptide bond with the last amino 2

Assignment 12

MBB 222 Summer 2020 (Craig)

acid added in the growing polypeptide chain; a tRNA for a specific amino acid has an anticodon that will base pair with a codon on mRNA specifying (coding for) that amino acid; in this way, tRNA “reads” the mRNA sequence (in the 5’-3’ direction) and translates that sequence into a protein sequence (N-terminal to C-terminal) (b) mRNA – messenger RNA - RNA molecule that encodes for protein sequences in units of codons (the message): one codon encodes one amino acid; mRNA sequence is read – translated - by tRNA (c) small subunit of the bacterial ribosome – 30S subunit, binds to mRNA and recognizes the ShineDelgarno sequence in bacterial mRNA, correctly positions the AUG start codon in its P-site for binding by the initiator tRNAfMet (d) large subunit of the bacterial ribosome - 50S subunit contributes to the A- and P-sites for binding an aminoacyl-tRNA (aa-tRNA) and the peptidyl tRNA (i.e. the tRNA to which the growing polypeptide chain is attached), respectively; also forms the E-site where the tRNA exits the ribosome; the 23S rRNA in the large subunit catalyzes the peptidyltransferase reaction (e) stop codon – UAG, UAA or UGA, do not encode for amino acids, signal termination of protein synthesis (f) transformylase (optional, FYI) – enzyme that adds a formyl group to Met after Met has been attached to the initiating tRNAfMet à฀ fMet-tRNAfMet (g) IF-2 (optional) – initiation factor 2, a GTPase that binds to GTP and to fMet-tRNAfMet and helps the tRNA to bind the P-site of the 30S subunit and base-pair with the start codon on mRNA (h) EF-Tu (optional) – elongation factor Tu, a GTPase that binds to GTP and to aa-tRNA and helps the tRNA to bind to the A-site on the 70S ribosome (i) RF1/2 (optional) – release factors 1 and 2; bind to the A-site at a stop codon and trigger dissociation of the translation machinery 7. How does the initiating bacterial AA-tRNAAA differ from other AA-tRNAAAs? Where does it bind on the ribosome? Is this the same binding site as other AA-tRNAAAs? The initiating bacterial AA-tRNAAA is fMet-tRNAfMet, which is a tRNA attached to a methionine modified by a formyl group (-COH). fMet-tRNAfMet binds to the start codon on the mRNA at the Psite of the small (30S) subunit of the ribosome (after which the large subunit binds to form the 70S initiation complex). All other AA-tRNAAAs bind at the A-site of the 70S complex. 8. Describe the peptidyltransferase step in the elongation phase of protein synthesis in bacteria. The incoming amino acid in the A-site is placed in the 50S peptidyltransferase site along with the terminal (last) amino acid on the growing polypeptide chain, which is attached to its tRNA in the Psite. The amino nitrogen of the incoming AA nucleophilically attacks the carbonyl carbon in the ester bond between the terminal amino acid and its tRNA. The polypeptide is then transferred to the incoming amino acid forming a new peptide bond. (This reaction in the peptidyltransferase site is catalyzed by the 23S ribozyme.) The discharged AA moves from the P- to the E-site and the peptidyl tRNA moves from the A- to the P-site. (Think about the ribosome moving along the mRNA in the 5- to 3’ direction. The tRNAs are base-paired with their codons and remain attached as the ribosome moves, shifting the tRNAs along the ribosome. Once in the E-site the discharged tRNA is released.)

3

Assignment 12

MBB 222 Summer 2020 (Craig)

9. Name a post-translational modification of proteins that we have discussed recently and explain its effect on the protein. Acetylation of N-terminal lysine side chains on histones; neutralizes the lysines so that they no longer attract DNA; loosens up the chromatin packaging; 30 nm fiber à฀ 11 nm fiber; allows replication/transcription machinery to access the DNA. 10. Why do cells have such an elaborate and efficient machinery for destruction of misfolded proteins? (optional) Because misfolded proteins can form aggregates via interaction of exposed regions. These aggregates can disrupt cell functions and cause cell death (some can form amyloid fibrils) so they must be dealt with immediately.

4...