Aris Kaksis Riga University docent - Chemistry

Biochemistry of Medicinals I Phar 6151 CHAPTER FIVE
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
Required reading: Stryer 4th edition Ch. 34, p. 875-888 and Ch. 33 p. 849-850

Protein Synthesis: Transfer RNA
I.          The Genetic Code
     In order for the sequence of mRNA to be translated into protein, the message must be deciphered into an amino acid AA sequence.
•     Translation depends on the concept three 3 bases (codon)-one 1 amino acid AA. There are 64 condons that correspond to 20 amino acids AA and 3 termination signals. AGU C

	U	C	A	G
U	UUU-Phe UUC-Phe UUA-Leu UUG-Leu	UCU-Ser UCC-Ser UCA-Ser UCG-Ser	UAU-Tyr UAC-Tyr UAA-Term UAG-Term	UGU-Cys UGC-Cys UGA-Term-SelenoCys-Trp-Mitochondria UGG-Trp
C	CUU-Leu CUC-Leu CUA-Leu CUG-Leu	CCU-Pro CCC-Pro CCA-Pro CCG-Pro	CAU-His CAC-His CAA-Gln CAG-Gln	CGU-Arg CGC-Arg CGA-Arg CGG-Arg
A	AUU-Ile AUC-Ile AUA-Ile AUG-Met	ACU-Thr ACC-Thr ACA-Thr ACG-Thr	AAU-Asn AAC-Asn AAA-Lys AAG-Lys	AGU-Ser AGC-Ser AGA-Arg AGG-Arg
G	GU-Val GUC-Val GUA-Val GUG-Val	GCU-Ala GCC-Ala GCA-Ala GCG-Ala	GAU-Asp GAC-Asp GAA-Glu GAG-Glu	GGU-Gly GGC-Gly GGA-Gly GGG-Gly

Consequently, the lanquage is degenerate. The degenerancy minimizes the effects of possible mutations. For example, if there was a transition in which T T C was changed to T T T, phenylalanine would still be incorporated into the growing polypeptide AA sequence.

•     DNA sequences are read form left to right, 5'--->3' or amino N-terminus to ---> carboxy C-terminus.

DNA         5'-A T G-GCC- T T T -GA T - T C T -AAA- T AA-3' (CODING STRAND)
RNA           5’-AUG-GCC -UUU-GAU -UCU-AAA-UAA-3’
Protein      N-Met     Ala     Phe     Asp    Ser     Lys   Stop-C

•     The code is nearly universal (i.e., bacteria to humans). The one exception is in mitochondria, where there are some differences. For example, UGA encodes for a Termination signal for genomic transcripts,
              for human mitochondria, UGA encodes for Trp.

II.        tRNA

To read the sequence of mRNA and begin the process of transferring amino acids AA to the growing C end of a polypeptide chain, the adaptor molecule tRNA, which is a hybrid of RNA and an amino acid AA, is needed.

A.        Structure

The sequences of more than 100 tRNA's are known. All tRNA's have common structural motifs and can be written in a "cloverleaf" pattern.

                                                              Anti-Codon tRNA   - GTA anticodon
                                                                                   mRNA - CUT codon
Characteristics of tRNA's
            •           Single poly-nucleotide chain; 73-93 bases in length
            •           Contain unusual and methylated bases (ex. cytidine to 5-methylcytidine)
            •           5' end is mono-phosphorylated
            •           Base sequence at 3' end is CCA
            •           Half 1 / 2 of sequence is base paired
            •           TYC loop (ribo-thymidine—pseudo-uracil—cytosine)
            •           DHU loop (dihydro-uracil)
á            Variable extra arm
•           Anticodon loop; 5'-pyr-pyr-XYZ-mod. pur.-variable base-3'
            •           L-shaped
            •           Two 2 double helix segments
•           Unusual base interactions present in tertiary 3° interactions
            •           Anti-codon and amino acid AA attachment sites are far apart
            •           CCA terminus is not conformationally restricted

B.        Formation

tRNA's are synthesized by RNA polymerases. There are 60 genes for tRNA's from E.coli that are clustered at 25 sites, which are transcribed as multimeric precursors.

•           Primary transcript is cleaved by RNase P to the 5' end. In the presence of Mg2+ RNA of
                                                                                          RNase P is the catalytic domain.
            •           3'-end is processed by RNase D

   RNase P                                                               RNase P

III.    Activation of Amino Acids

Peptide bond formation in solution is highly ¯ disfavored due to the large thermodynamic conditions DG > 0 for this reaction. To over come this, the carboxylic acid group -COO- must be activated by conversion to a
tRNA-amino acid ester, or aminoacyl-tRNA. The aminoacetyl-tRNA is referred to a being charged with the amino acid AA. The generation of aminoacyl-tRNA's is a two 2 step process carried out by a family of enzymes called aminoacyl-tRNA synthetases.

A. Classes of aminoacyl-tRNA synthetases

At least one 1 amino-tRNA synthetase exists for each amino acid AA and in some cases several exist. They can be grouped into Class I and Class II

      •    Class I: Arg, Cys, Gln, Glu, Ile,   Leu, Met, Trp, Tyr, Val        (Generally the Larger Amino Acids)

      •   Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr       (Generally the smaller amino acids)

B. Overall Reaction                    Amino-acyl-AMP formation from ATP and amino acid AA

• The amino acid AA is activated by conversion of the free carboxylic acid to a mixed anhydride. Although stable the aminoacyl-AMP intermediate does not leave the enzyme.

• The amino acid AA is then transferred to the appropriate tRNA

C.                                               Proofreading

Aminoacyl-tRNA synthetases are highly selective for their amino acid AA and tRNA.

1.         Amino acid
The recognition of amino acids AA by aminoacyl-tRNA synthetases is generally based on the size of the amino acid AA side chain (residue).
• Acylation site rejects amino acids AA that are larger than the correct one because the binding site is too small.
•           Hydrolytic site destroys activated intermediates that are smaller than the right amino acid AA.

Example: Valine vs isoleucine (isoleucine has an extra methylene -CH2- group)
If valine is mistakenly activated by tRNAIle  coding for isoleucine, it is hydrolyzed preventing its incorporation in tRNAIle :

Large Acylation Site Smaller Hydrolytic Site Large Acylation Site Smaller Hydrolytic Site

<-- Difference -->

• Acylation and hydrolytic sites can also discriminate based on hydrophobic verses polar interactions.

Example: Valine vs threonine (difference is –OH in place of –CH3)
If threonine is mistakenly activated by tRNAVal coding for valine, it is hydrolyzed preventing its incorporation in tRNAVal :

Hydrophobic Acylation Site Polar Hydrolytic Site Hydrophobic Acylation Site Polar Hydrolytic Site

<--- Difference -->

                                    Val is preferred                                                                                Thr is preferred

•           Some aminoacyl-tRNA synthetases are capable of discrimination at the acylation step
(i.e., tyrosyl-tRNA synthetases) and do not have a hydrolytic site.

2.       tRNA

Recognition of the correct tRNA by the synthetase can occur by the following mechanisms:

• Specific recognition of the anticodon; ex. an change the anticodon for tRNAtrp to that for methionine and get good acylation by tRNAMet.
• Stem sequences can be crucial; ex. tRNAAla depends on GC at position 3:70 doesn't care what the codon is.
• Both the stem regions and anticodon are needed; ex. tRNAGln.

IV.     tRNA Anticodon-Codon Recognition                             Anticodon
To transcribe the mRNA, the tRNA's must recognize the codon.         3    2   1              3    2   1
This recognition does not depend on the amino acid AA                      3'-X -Y -Z -5'     3'-C -G -A -5'
conjugated to the tRNA. It is entirely based on the tRNA and is         5'-X'-Y'-Z'-3'     5'-G -C -U -3'
based on the fact that many tRNA's are capable of binding to                 1    2   3               1    2   3
                                                  several different codons,                                         Codon

•     Other wobble base pairings also can exist: G and U or C and U and A or G
•     The following rules apply;
1)   First 1st two 2 bases of a codon pair in the standard way; GºC, A=T
2)   First 1st base of anticodon determines
whether the tRNA is able to recognize, one 1, two 2 or three 3 codons;
C then G,
A then U,
U then A      or G,
G then U      or C    and
I    then U, C or A