Aris Kaksis Riga University docent - Chemistry

Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink "mailto:Trety001@umn.edu"» - 6-3432
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Prof. mailto:ariska@latnet.lv

Transcription Transcription Translation

Translation

Antisense therapy: a new generation of drugs?

Potential advantages of antisense drugs:
1.Specificity.
2.Facilitated drug development process.
3.Reduced side effects.

Problems associated with antisense drugs:

1. Delivery through cell membranes.
2. Degradation with nucleases.
3. Possibility of severe side effects in some individuals.

Figure 2. Therapeutic Nucleic Acids

Replacement of oxygen O by sulfur S in each phosphate group (above) makes the beckbone resistant to nucleases, which would otherwise cut the bridges. However, the macromolecule remains electrically charged, impeding its passage across cell membrane. The entire backbone can be replaced with a chain resambling protein (bottom). The result is nuclease-resistant and the bases retain correct alignment. However, this construct, too, does not readily cross cell membrane.

Vitravene (Isis Pharmaceuticals, 1998)
¤ used against cytomegalovirus (herpes infection on the eye) in AIDS patients
¤ Phospho-thiolate oligonucleotide: 5’-GCG

GCG-3’
¤ Mechanism: binds mRNA coding for the protein required for viral replication and causes its degradation
with RNAase H.

Protein Synthesis Part 1: Take Home Message

Required reading: Stryer Ch. 34, p. 875-888 Ch. 33 p. 849-850
1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid
            (AA codons).
2) The mRNA message is read by tRNA through the use of a three base complement to the three base word
            (anticodon).
3) A specific amino acid is conjugated to a specific tRNA.
4) Amino acid AA side chain size, hydro-phobicity and polarity govern the ability of tRNA synthetases
                                                          to conjugate a specific three base message with a specific amino acid AA.

How do we go from mRNA to Protein?

DNA ----> mRNA -------------> Proteine
   t-RNA ---->|| Amino Acid Sequence

Translating the Message

DNA           5'-A

G-GCC-

-GA

-AAA-

AA-3'
RNA 5'-A

G-GCC-

-GA

-AAA-

AA-3'
Protein      N-- met     ala      phe      asp       ser     lys     stop -C

Translation of mRNA

            Sequence of 3 Bases = One Codon
                            One Codon = One Amino Acid AA
20 Natural Amino Acids AA = 64 Codons

“Degenerate Code: Several Different Codons per Amino Acid AA”

Table 1. The genetic code. for mRNA Y                                                     Genetic Code

1st position	2nd position				3rd position
(5' end)-->		C	A	G	(3' end)-->
	Phe Phe Leu Leu	Ser Ser Ser Ser	Tyr Tyr STOP STOP	Cys Cys STOP SelenoCys Trp Mitochondria Trp	C A G
C	Leu Leu Leu Leu	Pro Pro Pro Pro	His His Gln Gln	Arg Arg Arg Arg	C A G
A	Ile Ile Ile Met init	Thr Thr Thr Thr	Asn Asn Lys Lys	Ser Ser Arg Arg	C A G
G	Val Val Val Val	Ala Ala Ala Ala	Asp Asp Glu Glu	Gly Gly Gly Gly	C A G

Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of protein synthesis according to the rules shown. The codons G

G and GAG, for example, are translated into valine and glutamic acid, respectively. Note that those codons with

or C as the second 2 nucleotide tend to specify the more hydrophobic amino acids AA.

How do we go from mRNA to Protein?

Incoming tRNA

Transfer RNA

• Acts as an adaptor molecule between mRNA and peptide sequence
• Contains amino acid AA attachment site and template recognition site

Structure of Transfer RNA

Di-hydro-uridine (DHU) Anti Codon Pseudo-uridine (Y)

T-RNA has an L Tertiary Structure

T Loop || || 5’ || 3’ Stem Acceptor

Anti-Codon

t-RNA Formation

Rnase P Rnase P

Classes of Aminoacyl-tRNA Synthetases

• 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)
                                                                        Charged tRNA -76

tRNA Activation by aminoacyl tRNA synthases
1. Amino-acyl-AMP formation

2. Aminoacyl transfer to the appropriate tRNA

tRNA Synthetase Proofreading Ile
Large Smaller Large Smaller
Acylation Site Hydrolytic Site Acylation Site Hydrolytic Site

¬ Difference ®

Correct Acylation                                                                                                                         Mis-acylation

tRNA Synthetase Proofreading Val

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

¬ Difference ®

Correct Acylation                                                                                                                  Mis-acylation
tRNA Recognition by Synthetases

¤     Specific recognition of the anticodon; ex. an change the anticodon for tRNA-Trp to that for methionine and get good acylation by tRNA-Met.

¤     Stem sequences can be crucial; ex. TRNA-Ala depends on GC at position 3:70 doesn't care what the codon is.
¤     Both the stem regions and anticodon are needed; ex. TRNA-Gln.

tRNA Recognition by Synthetases

Anticodon Recognition
Table 1. The genetic code. for mRNA Y Genetic Code

1st position	2nd position				3rd position
(5' end)-->		C	A	G	(3' end)-->
	Phe Phe Leu Leu	Ser Ser Ser Ser	Tyr Tyr STOP STOP	Cys Cys STOP SelenoCys Trp Mitochondria Trp	C A G
C	Leu Leu Leu Leu	Pro Pro Pro Pro	His His Gln Gln	Arg Arg Arg Arg	C A G
A	Ile Ile Ile Met init	Thr Thr Thr Thr	Asn Asn Lys Lys	Ser Ser Arg Arg	C A G
G	Val Val Val Val	Ala Ala Ala Ala	Asp Asp Glu Glu	Gly Gly Gly Gly	C A G

Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of protein synthesis according to the rules shown. The codons G

G and GAG, for example, are translated into valine and glutamic acid, respectively. Note that those codons with

or C as the second 2 nucleotide tend to specify the more hydrophobic amino acids AA.

Codon : Anticodon

First 1st Base of Anticodon is Variable
||
                   3 2 1
t RNA- 3'-X Y Z-5' anticodon
mRNA- 5'-X’Y’Z’-3' codon
                   1 2 3

Third 3rd Base of Codon is Variable

tRNA Anticodon-Codon Recognition

              Adenosine                                Inosine                                                      Guanosine

Anticodon 3’ – C – G – I – 5’ 3’ – C – G – I – 5’ 3’ – C – G – I – 5’
Codon 5’ – C – G – C – 3’ 5’ – C – G – A – 3’ 5’ – C – G –

– 3’

I------C base pair I-----A base pair I-----

base pair

tRNA Anticodon-Codon Recognition

Anticodon 3’ – C – G – I – 5’ 3’ – C – G – I – 5’ 3’ – C – G – I – 5’
Codon 5’ – C – G –

– 3’               5’ – C – G – C – 3’                   5’ – C – G – A – 3’

Anticodon 3’ – C – G – G – 5’               3’ – C – G – G – 5’
Codon       5’ – C – G –

– 3’ 5’ – C – G – C – 3’

Anticodon 3’ – C – G –

– 5’ 3’ – C – G –

– 5’
Codon       5’ – C – G – A – 3’               5’ – C – G – G – 3’

Anticodon 3’ – C – G – C – 5’               3’ – C – G – A – 5’
Codon       5’ – C – G – G – 3’               5’ – C – G –

– 3’

Protein Synthesis Part 1: Take Home Message

1) Translation of the genetic code is dependent on three base words
                                                                                                   that correspond to a single amino acid.
2) The mRNA message is read by tRNA through the use of a three base complement to the
                                                                                                                                              three base word.
3) A specific amino acid is conjugated to a specific tRNA (three base word).
4) Amino acid side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases
                                       to conjugate a specific three base message with a specific amino acid.