Lecture - 2-tRNA Structure & Function
Lecture - 2-tRNA Structure & Function
Lecture - 2-tRNA Structure & Function
1)
mRNA
Messenger RNA, a copy of DNA blueprint of the gene to be expressed. Aminoacyl transfer RNA, also called anticodon or adaptor molecule. One or more tRNAs for each amino acid. A very large complex of several rRNAs (ribosomal RNA) and many protein molecules. Total molecular weight over 2 million dalton.
Information
tRNA
Supply
Ribosome
Factory
Protein
Polypeptide chain with sequence dictated by the mRNA sequence. Also called the gene product.
Product
Protein Synthesis
Electronmicrograph of a so-called polysome: one mRNA strand (faint horizontal line) with many individual ribosomes attached (dark blobs). The newly synthesized polypeptide chains (proteins) can be seen as irregularly shaped extensions from the ribosomes:
The bottom panel shows a schematic representation of the process in the upper panel.
Now let's magnify a ribosome to the size of a "Big Mac" (factor 10,000,000). At this magnification an E. coli bacterium would
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be about 10 meters (or 30 feet) in diameter. You would be about 10,000 miles tall:
Ribosome
Big Mac 20 cm 5 cm
8 inches
tRNA
2 inches
mRNA (900 bases) Extended (unfolded) protein (300 amino acids) Globular (folded) protein (300 amino acids)
450 cm
15 feet
90 cm
3 feet
5 cm
2 inches
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tRNA Structure
Primary & secondary structure
q
All tRNAs have similar sequences of 73 to 93 nucleotides 3' end always terminates with the sequence CCA, with the 3' hydroxyl of the ribose of the terminal A being the point of covalent attachment of the amino acid Contain a number (7-15%) of unique/modified bases. These are post-transcriptionally modified after synthesis by RNA polymerase. In particular, adenosine (A) in first or 5' position of the anticodon (corresponding to the third or 3' position of the codon) is always modified to inosine (I) which lacks the amino group on the
r
q q
purine ring. Inosine can base-pair with A, U or C and thus accounts for much of the degeneracy of the Genetic Code ("Wobble Theory"). tRNAs have cloverleaf secondary structure due to four base-paired stems The cloverleaf contains three non-base-paired loops: D, anticodon, and TpsiC loop
Tertiary structure
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q q q q
The tertiary structure of tRNA is best described as a compact "L" shape. The anticodon is a singlestranded loop at the bottom of the Figure which later basepairs with the triplet codon The amino acid is attached to the terminal A on the upper right. The active sites (anticodon and amino acid) are maximally separated. As in proteins, the tertiary structure is dictated by the primary sequence. The tertiary structure is stabilized by base pairing and base stacking. Two areas (anticodon stem and acceptor stem) form double helix.
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Each tRNA is charged with the proper amino acid via a covalent ester bond at their 3' end by a family of enzymes called aminoacyl-tRNA synthetases. Each enzyme must recognize both the tRNA specific for an amino acid and the corresponding amino acid. This energyconsuming process is ATP-dependent and results in the cleavage of two high-energy phosphate bonds (one in going from ATP to AMP + PP, and one for the cleavage of pyrophosphate into two inorganic phosphates:
There are 20 different aminoacyl-tRNA synthetases, one for each amino acid. Despite the fact that they all carry out very similar tasks, they vary greatly in size (40-100 kDalton).
Since there are 61 amino acid codons, most tRNA synthetases must be able to recognize more than one type of tRNA (i.e. 6 codons for Arg). These tRNAs are called cognate tRNAs for that particular synthetase. This mapping is achieved through so-called recognition domains on the tRNA. tRNA shown with red backbone and yellow bases. tRNA synthetase shown as space-filling model in blue:
The recognition domain includes unique of sections of the acceptor stem and/or the anticodon (black dots):
The accuracy of charging tRNA with the proper amino acid is crucial because once charged, only the tRNA anticodon determines incorporation, not the attached amino acid.
The error rate of charging is very low: 1 in 10,000. This is achieved by two means:
the amino acid specificity pocket in a specific synthetase will only bind amino acids similar in size and charge. the synthetase also has
proofreading capability which, once a wrong aminoacyladenylate complex is formed (1st step), will hydrolyze the complex before it can be covalently attached to the tRNA (2nd step).
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Assumption: Each tRNA, defined by its 3-base anticodon, pairs only with its complementary codon on the mRNA.
Discrepancy: tRNAAla was found to bind to codons GCA, GCC and GCU.
GCA CGU
GCC CGG
GCU CGA
The answer: The tRNAAla anticodon is actually CGI which pairs with all three codons.
The first two positions of the mRNA codon observe Watson-Crick base pairing rules (A-U, C-G) The third position exhibits wobble. Wobble occurs because the conformation of the tRNA anticodon loop permits flexibility at the first base of the anticodon.
q q
(not observed)
U G
(Watson-Crick) (Watson-Crick)
C G U
C A
or or
U G
or
or
Caveat: Since a single tRNA can respond to more than one codon, one tRNA could respond to codons for two different amino acids! This would lead to an ambiguous code. But since the Genetic Code is unambiguous, certain anticodons are disallowed.
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One anticodon for serine is UCG: Codon (5'->3') Anticodon (3'->5') Amino acid AGC UCG Ser AGU UCG Ser
(wobble)
Now, in the following hypothetical example, anticodon UCA (if it existed) would also base-pair with codon AGU using wobble rules: AGU AGU UCA => UCI Ser Ser AGC UCI Ser AGU UCI Ser AGA UCI Arg
(wobble)
(wobble)
But anticodon UCA would be converted post-transcriptionally to UCI which is able to bind three codons (wobble rules), including one for Arg! For this reason the UCA/UCI anticodon does not exist! And the codon AGA for Arg is actually covered by the anticodon UCU (wobble)!
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Supplemental Material
This page won't be on the final!
Molecular Graphics: This exercise will let you get a better 3-dimensional picture of the interaction of a tRNA with its tRNA synthetase. You will be able to rotate, scale and otherwise manipulate the structure on your screen.
First, you'll need to obtain & install a FREE molecular graphics program called RASMOL on your PC:
Unpack the distribution, you should then be able to run the program RASWIN (PC) or RASMAC (MAC). Click here for detailed help. If you get stuck or need help, please contact me (Hudel). Second, 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 as "1GTR.pdb". This file contains the complete atomic coordinates of GLUTAMINYL-tRNA SYNTHETASE COMPLEXED WITH tRNA AND ATP.
Now, run the program RASWIN/RASMAC and go to the "Open..." option under the "File" Menu. Select the file you just saved (1GTR.pdb). Then select the "Backbone" option under the "Display" menu. Your display should show something like this. For a description of the program, check the "User Manual" under the "Help" Menu.
Have fun!
Next: Summary
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Summary (2.7)
Summary
Features of tRNAs
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tRNAs are the adaptor molecules that translate from nucleic acid triplet to amino acid. tRNAs are charged with amino acids by enzymes called aminoacyltRNA synthetases in an energy-dependent 2-step process. There are 20 specific synthetases, one for each amino acid. Since most amino acids have more than one possible codon, there are more than 20 different tRNAs (40 in E. coli). Not all 61 codons have a specific tRNA due to wobble in the 3rd codon position. tRNA synthetases highly specific and have proofreading capabilities, achieving a better than 1:10,000 error rate. The accuracy of charging tRNA with the proper amino acid is crucial because once charged, only the tRNA anticodon determines incorporation into the growing polypeptide chain, not the attached amino acid.
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