aaRS and Ribosome Structure

Question 1

What recognition steps does translation require? What are they mediated by?

Answer 1

At least two steps:
-Choice of correct amino acid for covalent attachment to tRNA (mediated by aminoacyl-tRNA synthetases (aaRS)
-Selection of an amino acid-charged tRNA specified by the mRNA (mediated by ribosome)
-Isoaccepting tRNAs are a challenge for both

Question 2

Aminoacyl-tRNA synthetases (aaRS)

Answer 2

-Choice of correct amino acid for covalent attachment to tRNA is catalyzed by amino acid-specific enzyme, aaRS
-Append an amino acid to the 3’ end of a cognate tRNA
-Unfavorable process, requires hydrolysis of 1 ATP and 2 sequential reactions

Question 3

How many different aaRS are there?

Answer 3

20

Question 4

What is charging of a tRNA?

Answer 4

-When an amino acid is added to the 3’ OH of the tRNA

Question 5

What is the reaction mechanism for adding amino acids to tRNAs? Where do the steps occur?

Answer 5

-Step 1: activation of the amino acid (generate aminoacyl-AMP) (enzyme bound intermediate)
—–amino acid + ATP = aminoacyl-AMP + PPi
-Step 2: transfer of the amino acid to the tRNA (generate aminoacyl-tRNA) (MECHANISM DEPENDS ON ENZYME CLASS)
—–aminoacyl-AMP + tRNA = aminoacyl-tRNA + AMP
-Overall: amino acid + tRNA + ATP = aminoacyl-tRNA + AMP + PPi
-Both steps occur in the active site of the enzyme with no dissociation of the intermediate state
-Diagram L17 pg9

Question 6

Class I aaRS

Answer 6

-Attach amino acid to 2’-OH then transesterify to 3’-OH
-Requires anticodon recognition to aminoacylate their tRNA
-Monomers

Question 7

Class II aaRS

Answer 7

-Attach amino acid immediately to 3’-OH
-Do not interact with anticodon of tRNA
-Homodimers

Question 8

What do class I and II of aaRS have in common? What about evolution?

Answer 8

Both modify the 3’ adenosine of tRNA but are likely unrelated

Question 9

What is the second genetic code?

Answer 9

aaRS recognizing the tRNAs that code for their amino acids, must be very specific

Question 10

Where are the nucleotides in the tRNA that confer binding specificity with aaRS concentrated in?

Answer 10

-Acceptor stem
-Anticodon arm
-Nucleotides of the anticodon itself
-These nucleotides are called tRNA identity elements

Question 11

What is proofreading based on in aaRS?

Answer 11

-Based on size and hydrophilicity

Question 12

What is specificity the result of in aaRS?

Answer 12

-Product of synthesis and proofreading steps
-Aminoacylated tRNAs visit editing/proofreading site of the enzyme before dissociating

Question 13

tRNA^Thr proofreading basics

Answer 13

-Threonine is similar to Valine in size and serine in hydrophobicity
-Activation domain uses hydrophobicity to select for amino acids
-Editing uses size to select for amino acids

Question 14

Synthetic/activation domain of threonyl-tRNA synthetase

Answer 14

-Uses hydrophobicity
-Contains a zinc ion at the active site which binds to the amino and hydroxyl groups of Thr
-Used to avoid coupling incorrect amino acid
-Val is similar in structure to Thr, but lacks hydroxyl group, so it is not joined to tRNA^Thr
-Results in Ser occasionally linking to tRNA^Thr due to hydroxyl group

Question 15

Editing domain tRNA^Thr

Answer 15

-Threonyl-tRNA synthetase has an editing site that hydrolyzes Ser-tRNA
-Thr is sterically excluded from editing site due to extra methyl group
-Aminoacylated CCA arm can swing out of the activation site and into editing site to remove Ser
-Determined by size
-Most aminoacyl-tRNA synthetases contain editing sites and activation sites to ensure very high fidelity

Question 16

What is a ribosome (prokaryote specifics)

Answer 16

-Amino acid polymerase
-2/3 RNA, 1/3 protein
-Big complex
-E. coli = 70S
-Contains 30S and 50S subunits
-Self-assemble when all parts available

Question 17

Why do the 30S and 50S subunits of ribosome not add up to 70S

Answer 17

-This is based on sedimentation, which is the ratio of speed (in a centrifuge) to acceleration
-Does not add the same way as weight and other stuff

Question 18

30S structure

Answer 18

-Mitten-shaped
-16S rRNA
-21 proteins

Question 19

50S

Answer 19

-Large spheroidal
-3 proturbances
-23S rRNA
-5S rRNA
-32 proteins

Question 20

Ribosomal proteins

Answer 20

-Designated with S or L for subunit, followed by number indicating position on a gel in decreasing mass
-Only one protein is common in both subunits
-Contain RNA recognition motif (RRM) which occurs in many RNA-binding proteins

Question 21

Ribosome structure resolution

Answer 21

-Revealed by cryo-EM
-Freezes so rapidly that water doesn’t crystallize
-Sample remains hydrated and in native shape

Question 22

How does the ribosome work with structure

Answer 22

-Between the two subunits is where tRNA and mRNA bind
-Small subunit recognizes mRNA and tRNA
-Large subunit catalyzes reaction of polypeptide elongation
-tRNA anticodon loop is bound to 30S subunit, rest is bound to 50S
-Interactions are mainly RNA-RNA
-rRNA binds to tRNA’s conserved segments, permitting ribosome to bind different tRNAs in similar ways

Question 23

Ribosome overall structure all together

Answer 23

-30S and 50S subunits form a cleft through which mRNA passes
-No protein within 18A of the active site for peptide bond fomation, means that protein is mainly scaffolding
-Ribosome is a ribozyme
-Bacterial ribosomes can be stripped of most protein and still retain ability to catalyze peptide-bond formaiton