Aris Kaksis Riga University docent - Chemistry

Protein Synthesis Part 3: Take Home Message
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
Required reading: Stryer 4th Ed. Ch. 34 p. 888-908
1) Translation always proceeds by reading the mRNA sequence from the 5’ to the 3’ end.
2) Amino acids AA are assembled in to proteins with the first amino acid AA1 having a free amino group
(N-terminus) –NH2 and the last having a free carboxylate –COO- (C-terminus) (N®C)
3) Synthesis of the protein is processive and contains 3 steps;
a) initiation, b) elongation and c) termination

Overall Ribosome Structure: Translation Machine

70S

Large Subunit S50

Small Subunit S30

Prokaryotic Ribosomes 70S
Two tRNA binding sites

Peptidyl || transferase
ridge || ||GTPase back
50 S

Exit site Prokaryotic Ribosomes 70S

Tertiary 3° structure of 16S rRNA

Overview of Translation in prokaryotes

Several ribosomes simultaneously translate a messenger RNA

Five 5 and 35 ribosomes read the same mRNA sequentially
Growing polypeptide chaines Complete polypeptide

5’ initiator codon A

                                     Ribosome movement ------------------------>

Translating the Message

DNA           5'-ATG-GCC-TTT-GAT-TCT-AAA-TAA-3'

mRNA        5'-AUG-GCC-UUU-GAU-UCU-AAA-UAA-3'

Protein      N- Met   Ala   Phe   Asp   Ser    Lys Stop –C

Write the sequence in a polypeptide translated from the folowing mRNA:

RNA           5'-GGA-GGA-G

A-AG

-3'

Protein N- Gly -Gly - Val - Ser - Cys –C

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 td>
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.

Prokaryotic mRNAs often are polycistronic (encode more than one polypeptide)

(a) transcription in the presence of an inducer

RNA polymerase binds, transcription of structural genees begins
Inactivated repressor cannot bind to operator
Inducer molecules bind to repressor, inactivating it
Beta-Galactosidase Permease Acetyl transferase

Translation: Formyl Methionine

                                                          tRNAf Initiator tRNA

Initiation of Translation: Ribosomal Binding Site

                                             Shine-Delgarno
16S

CCA
mRNA 5’ GA

AGG AGG

GACC

G GCC

3’
Protein fMet Ala Phe

Initiating AA tRNA is Met, not fMet;
Initiator tRNA is Met-tRNA;
Initiatior signal is AUG – no Shine-Delgarno sequence;
40S subunit-Met-tRNA; -initiation factors complex binds 5’ cap and scans mRNA for the nearest A

G

Protein synthesis: Termination

When the ribosome encounters a stop codon (shown as the triplet), there is no tRNA attracted and the ribosome separates and leaves the mRNA.
5’

3’ 5’
Stop codon Stop codon

tRNA binding sites inside the E. coli Ribosome

large subunit tRNA tRNA

Small subunit mRNA location

Large ribosome subunit: PDB 1FFK Large Ribosome Subunit

                                                                   Crown”view with RNAs blue, in spacefill; proteins red, as bacjbone.

                                                  Protein factors involved in translation
Initiation                                 Role
IF 1, 3             Assembly of initiation complex
IF 2                 Brings initiator fMet-tRNAf

Elongation
EF-Tu              Carries tRNAs to the A ribosomal site
EF-Ts             Interchange EF-Tu with GTP
EF-G               Translocation

Termination
RF-1                Release factor (

AA,

AG)
RF-2 Release factor (

AA,

GA)
RF-3 GTPase, promotes release

Initiation of protein synthesis

Ribosomal Binding Site

30S

30S•IF1•IF2•IF3

fMet fMet
5’

mRNA

5’

mRNA
S.D.

Protein synthesis: Elongation
Large ribosome binds and then the next tRNA binds at A site
Peptide bond forms: complex then moves to P site
Another tRNA moves into the A site in order to add another amino acid AA to the peptide chain NH3+

Transfer of aa-tRNA to Ribosome

[Tu] ®®® [AA-tRNA]

                         Peptidyl Transferase
                          P                                                                         A

Translocation
NH3+.

Ribosome Synthesis of Polypeptide
A = Acceptor
P = Product
E = Exit

E P A E P A

E P A E P A E P A

¯¾¾® Continued Peptide Synthesis

Termination of Peptide Synthesis by RF1 and RF2

E P A

<---

C

Termination of Translation

mRNA 5'

Ribosome t-RNA Binding Sites

A Steitz, 2000 B

Ribosome Peptidyl Transferase Steitz, 2000

A B

C D

E F

Protein synthesis as target for drug design

• Can target various steps in protein synthesis: initiation, elongation, translocation
• Many natural antibiotics – leads for drug design

• mechanisms of selective toxicity:
Selective permeability of bacterial cell wall and membranes
Differences in structure and mechanism of bacterial protein synthesis and eukaryotes – can target bacteria

• mechanisms of resistance:
Lowered ¯ permeability of cytoplasmic membrane
Change in shape of the ribosomal binding site
Enzymic modification by a plasmid-borne gene
Drugs that Target Translation

Antibiotics interfere with the processes of translation by several different mechanisms.