27: Proteins

Question 1

how is mRNA information linked to specific amino acids?

Answer 1

every three bases codes for an amino acid (codon) within a reading frame. the code is non-overlapping (mostly). final product can be altered by small mutations if they cause shifts in the reading frame

Question 2

What is translational frameshifting? examples

Answer 2

programmed change in the reading frame during translation of an mRNA on a ribosome, occurring by any of several mechanisms. Example: product of the pol gene (RT) is translated as a larger poly protein on the same mRNA as Gag protein. production requires a translational frameshift in an overlap region to bypass a stop codon in the Gag gene. the Gag-Pol protein is trimmed to the mature RT by digestion.

Question 3

what is RNA editing? examples

Answer 3

post translational modification of an mRNA that alters the meaning of one or more codons during translation. Includes addition/deletion of nucleotides, which require a special class of guide RNA molecules that act as templates in the editing process. editing can also include alteration of nucleotides like deamination of A or C, uses ADARs and APOBEC enzymes

Question 4

common characteristics of the amino acid code. what are the initiator and termination codons

Answer 4

Code is degenerate, universal (mtDNA can have variations and rarely stop codons are adapted to encode unusual aa’s), and has initiator and terminator codons:
AUG

UAA
UAG
UGA

Question 5

define open reading frame

Answer 5

ORF starts at a start codon AUG, is generally 50 consecutive codons, and ends in termination codon

Question 6

overview of protein translation in E coli

Answer 6

tRNA is aminoacylated (charged). mRNA and charged tRNA bind to the small ribosomal subunit. the large subunit then binds. elongation proceeds in successive cycles of aminoacyl tRNA binding and peptide bond formation until the ribosome reaches a stop codon (mRNA is read 5’ - 3’, built Nt - Ct direction). Translation stops when a stop codon is encountered. mRNA and protein dissociate and ribosome subunits recycled. protein folds and is posttranslationally processed
slide 8

Question 7

structure of tRNAs

Answer 7

four arms, two of particular note: anticodon arm contains the anticodon which pairs with the mRNA codon and ‘reads’ the code. amino acid arm which attaches to the appropriate amino acid and carries it for peptide synthesis. amino acid is attached via aminoacyl group
draw general structure slide 11

Question 8

mech for activation of amino acids (tRNA charging)

Answer 8

slide 12
1. adenylate the amino acid
2. attach amino acid to tRNA via aminoacyl-tRNA synthetase. class I uses 2’ OH as nucleophile, class II uses 3’ OH as nucleophile. both have same end product of 3’ aminoacyl group
NET: amino acid + tRNA + ATP –> aminoacyl-tRNA + AMP

Question 9

how does tRNA synthetase show selectivity

Answer 9

two filters:
1. bind correct amino acid and catalyze formation of the aminoacyl adenylate
2. bind incorrect aminoacyl adenylate at a second site, results in hydrolysis
proofreading occurs after formation of activated aa but before covalent attachment to tRNA. synthetases can hydrolyze an incorrect amino acid from the tRNA
Often just a single pair in the tRNA is required for recognition by the synthetase (slide 14)

Question 10

what are ribosomes composed of

Answer 10

rRNA and proteins (mostly RNA). the large and small subunits are 65% rRNA and 35% protein. large subunit has three sites for tRNAs, A, P, and E. small subunit has site for the part of mRNA that will interact with anticodon. The active site is composed of RNA! not protein

Question 11

describe the components of initiation of translation

Answer 11

components: 30S, mRNA, fMet (bacteria), initiation factors, GTP, 50S, Mg2+. All organisms have TWO tRNAs for Met, one is used exclusively when AUG is the initiator codon!
Bacteria: N-formylmethionine (fMet) which has no way of being used internally due to inactive amino group. Eukaryotes: iMet - but fMet in mitochondria
draw fMet. slide 20

Question 12

process of initiation of translation

Answer 12

initiation factors bind 30S subunit. Shine-dalgarno sequence pairs with 16S rRNA and positions AUG start site in the correct place. IF2-GTP binds and recruits fMet-tRNA which pairs with start codon. 50S subunit associates, IF2 hydrolyzes GTP initiation factors leave. Complex initiation complete

Question 13

describe elongation of translation in bacteria

Answer 13

initiator fMet-tRNA binds in P site. the 2nd incoming aminoacyl-tRNA binds in A site using elongation factors. peptide bond forms with amine of incoming aminoacyl-tRNA molecule acting as nucleophile. GTP hydrolysis of Tu (elongation factor) serves as checkpoint for correct tRNA and tight binding. Once bound, peptide is translocated (by Ef-G, a translocase) to shift and allow another aminoacyl-tRNA to enter A site
mech slide 23

Question 14

describe termination of translation in bacteria

Answer 14

Stop codon is encountered. termination factors contribute to hydrolysis of the terminal aminoacyl-tRNA. components dissociate, free tRNA, polypeptide, small and large subunits

Question 15

define polysome

Answer 15

a complex of an mRNA molecule and 2 or more ribosomes.
in bacteria, protein translation and DNA transcription are coupled and occur simultaneously via polysomes. eukaryotes have nuclei so this doesn’t happen

Question 16

what are protein translation energy requirements and how is this coupled to fidelity?

Answer 16

charging tRNA’s costs ATP, corrections need ATP, chaperones use GTP, overall the average is about 4 NTPs per peptide bond. rate of hydrolysis is coupled to fidelity because of Tu, the proofreading mech, that uses GTP hydrolysis as a system to lock in the correct tRNA

Question 17

how is translation different in Eukaryotes

Answer 17

both 5’ and 3’ ends of the mRNA are tied up in the complex (A tail used)
translation efficiency is a factor of how well all the components interact
no shine-dalgarno sequence, uses scanning mech to find AUG codon
not coupled to transcription

Question 18

how are polypeptides processed? what are the most common amino acid modifications?

Answer 18

polypeptides lose signal sequences, have covalent modifications, and formation of disulfides. Initiator Met is often removed, carbohydrates attached in ER, isopropyl groups attached, prosthetic groups and metals added.
Serine, Threonine, and Tyrosine are most commonly phosphorylated. Lysine is commonly methylated (1-3 methyls added)

Question 19

what are targeting sequences

Answer 19

specific amino acid sequences recognized by specific proteins that help target the protein to the correct location (orgnelle, cell, etc).

Question 20

how are proteins targeted to the ER

Answer 20

as the mRNA is translated, the signal sequence is produced and is recognized by SRP (signal recognition particle) which halts translation and directs the complex to the ER surface. Translocation complex directs growing peptide into the ER lumen, ATP/GTP directed. signal cleaves inside the ER lumen

Question 21

how are proteins glycosylated

Answer 21

glycosylation occurs in the ER, most commonly on Asn or Ser residues. involves anchoring in membrane with dolichol, synthesizing, translocating (flipping inside membrane), more synthesis and branching, and finally nucleophilic attack by Asn on peptide and release of final protein
slide 32

Question 22

how are proteins degraded in eukaryotes

Answer 22

ubiquitin is a small polypeptide that is linked to proteins via Lys residue to signal degradation in the proteasome. Process involves 3 enzymes passing the ubiquitin around until transferring to target protein. A single ubiquitin addition can be a normal signal, like phosphorylation, but multiple ubiquitins leads to degradation
slide 33

Question 23

what are nuclear localization sequences

Answer 23

NLSs indicate that a protein is a nuclear protein and should be transported to the nucleus. The signal leads to association of proteins and passage through the nuclear pore complex then dissociation of those proteins, leaving the nuclear proton in the nucleoplasm.