Lecture_2-tRNA Structure & Function

7/29/2019 Lecture_2-tRNA Structure & Function

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RNA: Structure & Function (2.1)

Lecture 2

tRNA: Structure & Function

Major players inprotein synthesis:mRNA, tRNA and the ribosome

mRNA

Messenger RNA, a copy ofDNA blueprint of thegene to beexpressed.

Information

tRNA

Aminoacyl transfer RNA, also

calledanticodon oradaptormolecule. One or more tRNAsfor each amino acid.

Supply

Ribosome

A very large complex of severalrRNAs (ribosomal RNA) andmany protein molecules. Total

molecular weight over 2 milliondalton.

Factory

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Protein

Polypeptide chain with sequencedictated by the mRNA sequence.Also called thegene product.

Product

Protein Synthesis

lectronmicrograph of a so-called polysome: one mRNA stranaint horizontal line) with many individual ribosomes attache

dark blobs). The newly synthesized polypeptidechainsproteins) can be seen as irregularly shaped extensions fromhe ribosomes:



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he bottom panel shows a schematic representation of therocess in the upper panel.

ow let's magnify a ribosome to the size of a "Big Mac" (facto0,000,000). At this magnification an E. colibacterium would



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e about 10 meters (or 30 feet) in diameter. You would bebout 10,000 miles tall:

Ribosome

20 nm or

200

Big Mac

20 cm 8 inches

tRNA5 nm or

50 5 cm 2 inches

mRNA (900 bases)450 nm or

4,500

450 cm 15 feet

Extended (unfolded)

protein

(300 amino acids)

90 nm or900

90 cm 3 feet

Globular (folded) protein

(300 amino acids)

5 nm or

50 5 cm 2 inches

1 nm (nanometer) is 10-9 me1 (angstrom) is 10-10 meters or 0.1

Next: tRNA Structure

ease report typos, errors etc. byEMAIL (mention the title of this page).

mailto:[email protected]:[email protected]


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RNA Structure (2.2)

tRNA Structure

Primary & secondary structure

q All tRNAs have similar sequencesof 73 to 93 nucleotides

q 3' end always terminates with the

sequence CCA, with the 3'hydroxyl of the ribose of the

terminalAbeing the point ofcovalent attachment of the aminoacid

q Contain a number (7-15%) ofunique/modified bases. These arepost-transcriptionally modified

after synthesis by RNApolymerase.

r In particular, adenosine (A)in first or 5' position of theanticodon (corresponding to the third or 3' position of the codon)

always modified to inosine (I) which lacks the amino group on

purine ring. Inosine can base-pair withA, U or C and thusaccounts for much of thedegeneracy of the Genetic Code("Wobble Theory").

q tRNAs havecloverleafsecondary structure due to four base-pairedsteq The cloverleaf contains three non-base-paired loops: D, anticodon, an

TpsiC loop

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http://bass.bio.uci.edu/~hudel/bs99a/lecture21/clover5.jpg


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RNA Structure (2.2)

Tertiary structure

q The tertiary structure of tRNA is best described as a compact "L" shapq Theanticodonis a single-strandedloop at

thebottom ofthe Figurewhichlater base-pairs withthe triplet

codonq The

aminoacid isattachedto theterminal

Aon theupper right.

q The active sites (anticodon and amino acid) are maximally separated.q As in proteins, the tertiary structure is dictated by the primary sequencq The tertiary structure is stabilized by base pairing and base stacking.q Two areas (anticodon stem and acceptor stem) form double helix.

http://bass.bio.uci.edu/~hudel/bs99a/lecture21/trna_tert.gif


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RNA Structure (2.2)

Next: tRNA Function




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RNA Function: Synthetases (2.3)

tRNA Function: Synthetases

q Each tRNA is chargedwith the proper amino acid via a covalent est

bond at their 3' end by a family of enzymes calledaminoacyl-tRNAsynthetases. Each enzyme must recognizeboth the tRNA specific fan amino acid and the corresponding amino acid. This energy-consuming process is ATP-dependent and results in the cleavage oftwo high-energy phosphate bonds (one in going from ATP to AMP PP, and one for the cleavage of pyrophosphate into two inorganicphosphates:



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q There are 20 different aminoacyl-tRNA synthetases, one for eachamino acid. Despite the fact that they all carry out very similar taskthey vary greatly in size (40-100 kDalton).

http://bass.bio.uci.edu/~hudel/bs99a/lecture21/trna_charge2.gif


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q Since there are 61 amino acid codons, most tRNA synthetases mustable to recognize more than one type of tRNA (i.e. 6 codons for ArgThese tRNAs are calledcognate tRNAs for that particular synthetasThis mapping is achieved through so-calledrecognition domains onthe tRNA. tRNA shown with red backbone and yellow bases. tRN

synthetase shown as space-filling model in blue:

http://bass.bio.uci.edu/~hudel/bs99a/lecture21/trna_synth_3d.gif


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q The recognition domain includes unique of sections of the acceptor

stem and/orthe anticodon (black dots):



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Accuracy & Proofreading

q The accuracy of charging tRNA with the proper amino acid is crucibecause once charged, only the tRNAanticodon determinesincorporation,not the attached amino acid.

q The error rate of charging is very low: 1 in 10,000. This is achieveby two means:

r the amino acidspecificity pocket in aspecific synthetase willonly bind amino acidssimilar in size andcharge.

r the synthetase also has



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proofreading capability which, once a wrong aminoacyl-adenylate complex is formed (1st step), will hydrolyze thecomplexbefore it can be covalently attached to the tRNA (2ndstep).

Next: The Wobble Theory




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The Wobble Theory (2.4)

The Wobble Theory

Assumption:

Each tRNA, defined by its 3-base anticodon, pairsonly with its complementary codon on the mRNA.

Discrepancy:

tRNAAla was found to bind to codons GCA, GCCand GCU.

Codon (5'->3')

Anticodon (3'->5')

GCA

CGU

GCC

CGG

GCU

CGA



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The answer:

The tRNAAla

anticodon is actually CGIwhich pairswith all three codons.

xperimentally determined codon-anticodon pairing rules:

q The first two positions of the mRNA codon observe Watson-Crick base pairing

rules (A-U,C-G)q The third position exhibits wobble.q Wobble occurs because the conformation of the tRNA anticodon loop permits

flexibility at the first base of the anticodon.

5' anticodon base

(tRNA)

3' codon base

(mRNA)

A(not observed) U (Watson-Crick)

C G (Watson-Crick)

G C orU

U AorG



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I AorCorU

Caveat: Since a single tRNA can respond to more thanone codon, one tRNA could respond to codons for twodifferentamino acids! This wouldlead to an ambiguouscode. But since the Genetic Code is unambiguous, certaanticodons are disallowed.

Next: The Dangers of Wobble




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The Dangers of Wobble (2.5)

The Dangers of Wobble

ince a single tRNA can respond to more than one codon, onRNA could in theoryrespond to codons for two different

mino acids:

xample:AGC andAGU both code for Ser. Their anticodon is UCG using wobble

les. But anticodon UCAwould also pair withAGU (Ser). This anticodon (UCA)

ould be converted post-transcriptionally toUCI (Hey, that's us!) which would

cognizeAGC andAGU as intended, but alsoAGAwhich codes for Arg -- not good,ould result in an ambiguous Genetic Code!

One anticodon for serine is UCG:

Codon (5'->3')

Anticodon (3'->5')

AGC

UCG

AGU

UCG (wobble)

Amino acid Ser Ser

ow, in the following hypotheticalexample, anticodon UCA(

existed)would also base-pair with codonAGU using wobblules:

AGU AGUUCA => UCI (wobble)

AGC AGU AGAUCI UCI UCI (wobble)

Ser Ser Ser Ser Arg

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The Dangers of Wobble (2.5)

ut anticodon UCAwould be converted post-transcriptionally

o UCI which is able to bind three codons (wobble rules),

ncluding one for Arg! For this reason the UCA/UCI

nticodon does not exist! And the codonAGAfor Arg isctually covered by the anticodon UCU (wobble)!

Next: Supplemental Material




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upplemental Material (2.6)

Supplemental Material

This page won't be on the final!

Molecular Graphics:

his exercise will let you get a better 3-dimensional picture of the

nteraction of a tRNA with its tRNA synthetase. You will be able to rotacale and otherwise manipulate the structure on your screen.

rst, you'll need to obtain & install aFREE molecular graphics program called RASMOL on your P

Click here to obtain the MS Windows/NT version

or

Click here to obtain the Apple Macintosh version

Unpack the distribution, you should then be able to run the program RASWIN (PC) or RASM(MAC). Click here for detailed help. If you get stuck or need help, please contact me (Hudel

econd, you'll need to get a copy of the atomic coordinates from the Protein Data Bank:

PDB entry 1GTR

Save this rather large file (0.5 MBytes) with the "Save full entry to disk" option. Save the file"1GTR.pdb". This file contains the complete atomic coordinates of GLUTAMINYL-tRNASYNTHETASE COMPLEXED WITH tRNA AND ATP.

http://xtal.pharm.nwu.edu/rasmol/rasmol2.exehttp://xtal.pharm.nwu.edu/rasmol/rasmol-mac.sea.hqxhttp://xtal.pharm.nwu.edu/rasmol/index.htmlmailto:[email protected]://www.rcsb.org/pdb/cgi/explore.cgi?job=download&pdbId=1GTR&page=&pid=13218959018743&opt=show&format=PDB&pre=1http://www.rcsb.org/pdb/cgi/explore.cgi?job=download&pdbId=1GTR&page=&pid=13218959018743&opt=show&format=PDB&pre=1mailto:[email protected]://xtal.pharm.nwu.edu/rasmol/index.htmlhttp://xtal.pharm.nwu.edu/rasmol/rasmol-mac.sea.hqxhttp://xtal.pharm.nwu.edu/rasmol/rasmol2.exe


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upplemental Material (2.6)

ow, run the program RASWIN/RASMAC and go to the "Open..." option under the "File" Menu.elect the file you just saved (1GTR.pdb). Then select the "Backbone" option under the "Display"enu. Your display should show something like this. For a description of the program, check the "Uanual" under the "Help" Menu.

Have fun!

Next: Summary


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ummary (2.7)

Summary

Features of tRNAs

q tRNAs are theadaptor molecules that translate from nucleic acidtriplet to amino acid.

q tRNAs arechargedwith amino acids by enzymes called aminoacyltRNA synthetases in an energy-dependent 2-step process.

q There are 20 specific synthetases, one for each amino acid.q Since most amino acids have more than one possible codon, there ar

more than 20 different tRNAs (40 inE. coli).q Not all 61 codons have a specific tRNA due to wobble in the 3rd

codon position.q tRNA synthetases highly specific and haveproofreading capabilitie

achieving a better than 1:10,000 error rate.q The accuracy of charging tRNA with the proper amino acid iscruci

because once charged, only the tRNAanticodon determinesincorporation into the growing polypeptide chain,not the attachedamino acid.

Next Lecture: The Ribosome, rRNA and mRNA

Please report typos, errors etc. byEMAIL (mention the title of this page).
http://bass.bio.uci.edu/~hudel/bs99a/lecture22/index.htmlmailto:[email protected]:[email protected]://bass.bio.uci.edu/~hudel/bs99a/lecture22/index.html

Lecture_2-tRNA Structure & Function

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