Biochemia 2008/09

TRANSLACJA

Messenger RNA (mRNA)

m

7

Gppp

Cap

5’

5’ untranslated region

AUG

initiation
codon

translated (coding) region

(AAAA)

n

poly(A)
tail

3’ untranslated region

UGA

termination
codon

3’

AAUAAA

Transfer RNA

• tRNA is the “adaptor” molecule in protein synthesis

• acceptor stem

• CCA-3’ terminus to which amino acid is coupled

• carries amino acid on terminal adenosine

•anticodon stem and anticodon loop

Amino acid activation
and aminoacyl tRNA synthetases

• aminoacyl tRNA synthetases are the enzymes that “charge” the tRNAs

• 20 amino acids

• one aminoacyl tRNA synthetase for each amino acid

• can be several different “isoacceptor” tRNAs for each amino acid

• all isoacceptor tRNAs for an amino acid use the same synthetase

each aminoacyl tRNA synthetase binds

• amino acid

• ATP

• isoacceptor tRNAs

H

2

N-C-C-OH

H

R

-

-

O

=

ATP

H

2

N-C-C-O-P-O-ribose-adenine

H

R

-

-

O

=

amino acid

adenylated (activated)

amino acid

PPi

uncharged tRNA

H

2

N-C-C-O

H

R

-

-

O

=

aminoacyl

(charged)

tRNA

AMP

3’

Amino acid activation

and

tRNA charging

The genetic code

• consists of 64 triplet codons (A, G, C, U) 4

3

= 64

• all codons are used in protein synthesis

• 20 amino acids

• 3 termination (stop) codons: UAA, UAG, UGA

• AUG (methionine) is the start codon (also used internally)

• multiple codons for a single amino acid =

degeneracy

• 5 amino acids are specified by the first two nucleotides only

• 3 additional amino acids (Arg, Leu, and Ser) are specified by

six different codons

The Genetic
Code

UUU
UUC
UUA
UUG

CUU
CUC
CUA
CUG

AUU
AUC
AUA

AUG

GUU
GUC
GUA
GUG

UCU
UCC
UCA
UCG

CCU
CCC
CCA
CCG

ACU
ACC
ACA
ACG

GCU
GCC
GCA
GCG

UAU
UAC

UAA
UAG

CAU
CAC
CAA
CAG

AAU
AAC
AAA
AAG

GAU
GAC
GAA
GAG

UGU
UGC

UGA

UGG

CGU
CGC
CGA
CGG

AGU
AGC
AGA
AGG

GGU
GGC
GGA
GGG

Phe

Leu

Leu

Val

Ile

Met

Ser

Pro

Thr

Ala

Tyr

Stop

His

Gln

Asn

Lys

Asp

Glu

Cys

Arg

Ser

Arg

Gly

Stop

Trp

Codon-anticodon interactions

• codon-anticodon base-pairing is antiparallel

• the third position in the codon is frequently degenerate

• one tRNA can interact with more than one codon (therefore 50 tRNAs)

• wobble rules

• C with G or I (inosine)

• A with U or I

• G with C or U

• U with A, G, or I

• I with C, U, or A

5’

3’

A U G

U A C

3’

5’ tRNA

met

mRNA

5’

3’

C U A
G

G A U

3’

5’ tRNA

leu

mRNA

wobble base

• one tRNA

leu

can read two

of the leucine codons

Wobble Interactions

Cechy kodu:

Zdegerowany, niedwuznaczny,

nie nakładający się, uniwersalny,

bezprzestankowy

Reading frame

• reading frame is determined by the AUG initiation codon

• every subsequent triplet is read as a codon until reaching a stop codon

...AGAGCGGA.

AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.

UGA.UCGAAUAAA...

MET.ALA.GLU.TRP.LEU.SER.MET.SER

• a frameshift mutation

...AGAGCGGA.AUG.GCA.GA .UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...

• the new reading frame results in the wrong amino acid sequence and

the formation of a truncated protein

...AGAGCGGA.

AUG.GCA.GAU.GGC.

UAA.GCAUGUCGUGAUCGAAUAAA...

MET.ALA.

ASP.GLY

mRNA

5’ cap

40S subunit

M

eIF2

AUG

Initiator tRNA bound to the

small ribosomal subunit with the

eukaryotic initiation factor-2 (eIF2)

Initiation of protein synthesis: mRNA binding

The small subunit finds the 5’ cap and

scans down the mRNA to the first AUG codon

mRNA

5’

40S subunit

M

eIF2

AUG

• the initiation codon is recognized

• eIF2 dissociates from the complex

• the large ribosomal subunit binds

60S subunit

mRNA

5’

M

AUG

• aminoacyl tRNA binds the A-site

• first peptide bond is formed

GCC

A

mRNA

5’

M

AUG

GCC

A

Ribosome structure

A

P PP

P

P

P

P

P

P-site

peptidyl tRNA site

A-site

aminoacyl tRNA site

mRNA

5’

Small subunit

Large subunit

Ribosome with bound tRNAs and mRNA

C

NH

2

CH

3

-S-CH

2

-CH

2

-CH

O=C

Peptide bond formation

• peptide bond formation is

catalyzed by

peptidyl transferase

•

peptidyl transferase is contained within

a sequence of 28S rRNA in the
large ribosomal subunit

;

• the energy for peptide bond formation

comes from the ATP used in tRNA charging

• peptide bond formation results in a shift

of the nascent peptide from the P-site
to the A-site

NH

2

CH

3

-S-CH

2

-CH

2

-CH

O=C
O

tRNA

NH

2

CH

3

-CH

O=C
O

tRNA

N

P-site

A-site

OH

tRNA

NH
CH

3

-CH

O=C
O

tRNA

P

UCA

P

P

P

P

P

UCA GCA GGG UAG

A

P

P

P

P

Elongation

GCA GGG UAG

• following peptide bond formation

the uncharged tRNA dissociates
from the P-site

• the ribosome shifts one codon along

the mRNA, moving peptidyl tRNA
from the A-site to the P-site; this
translocation requires the
elongation factor EF2

• the next aminoacyl tRNA then

binds within the A-site; this tRNA
binding requires the elongation
factor EF1

• energy for elongation is provided by

the hydrolysis of two GTPs:

• one for translocation

• one for aminoacyl tRNA binding

EF1

EF2

P

UCA GCA GGG UAG

P

P

P

P

Termination

• when translation reaches the stop

codon, a release factor (RF) binds
within the A-site, recognizing the
stop codon

• release factor catalyzes the hydrolysis

of the completed polypeptide from
the peptidyl tRNA, and the entire
complex dissociates

RF

P

UCA GCA GGG UAG

P

P

P

P

P

P

P

Składniki reakcji terminacji translacji

w komórkach eukariotycznych

The model of the steps in eukaryotic translation
initiation
and the roles of the factors.

Annu.Rev.Biochem. 73:657, 2004

Annu.Rev.Biochem. 73:657, 2004

The model for translation elongation in eukaryotes

Annu.Rev.Biochem. 73:657, 2004

The model for translation termination in eukaryotes

mod.: Molecular Cell 28, 721,
2007

Inicjacja translacji w komórkach eukariotycznych

Regulation of eIF4F activity via the sequestration of eIF4E by 4E-BP.
The protein kinases that regulate the phosphorylation state of 4E-BP
emanate from them TOR and S6 kinase pathways. In general, these
pathways are activated by conditions that favor growth and are
inactivated by stress conditions.

Annu. Rev. Biochem. 68:913, 1999

Genes & Dev. 15: 1593,
2001

Classical protein translation and mechanisms
of control.

TRENDS in Biochemical Sciences 28, 91,2003

5'- UTR = 5'-untranslated region

TRENDS in Biochemical Sciences 28, 91,2003

Multiple types of end-to-end interactions of RNA. Poly(A)-binding
protein (PABP)-mediated interaction of eukaryotic intiation factor
(eIF) 4G and the poly(A) tail in capped, polyadenylated human
mRNA.

The ribosome - recycling concept.
A possible function of mRNA circularization could in volve facilitation of a
direct recycling of ribosomes or ribosomal subunits, after termination at
the stop codon, back to the 5’ region of the same mRNA (indicated by
the green arrow). This speculative model is supported by the
observation of circular polyribosomes, as well as interactions between
PABP, initiation factors bound to the cap structure, and the translation
termination factore eRF3.

TIBS 28, 91, 2003

UTR = 5’- untranslated region

TIBS 28, 91, 2003

ORF (Open Reading Frame) - Otwarta ramka odczytu

Protein maturation: modification, secretion, targeting

5’

AUG

polysome for secreted protein

2. the signal recognition particle

a

(SRP)

binds the signal peptide

b

and

halts translation

1. translation initiates as usual
on a cytosolic mRNA

a

the

signal recognition particle (SRP)

consists of protein and RNA (7SL RNA); it binds

to the signal peptide, to the ribosome, and to the SRP receptor on the ER membrane

b

the signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus

of a protein, that consists mostly of hydrophobic amino acids

c

ER = endoplasmic reticulum

ER

lumen

c

cytosol

3. the SRP docks with the SRP receptor on
the cytosolic side of the ER membrane
and positions the signal peptide for
insertion through a pore

SRP

SRP receptor

Translation of a secreted protein

5’

ER lumen

cytosol

4. translation resumes and the nascent
polypeptide moves into the ER lumen

5.

signal peptidase

, which is in the ER

lumen, cleaves off the signal peptide

7. the ribosomes dock onto the
ER membrane; the rough ER
is ER studded with polysomes

6. the SRP is released
and is recycled

5’

ER lumen

cytosol

UGA

8. translation continues with the nascent
polypeptide emerging into the ER lumen

9. at termination of translation, the completed protein is
within the ER and is further processed prior to secretion

completed
protein is
processed and
secreted

Annu.Rev.Biochem. 73:539, 2004

• Examples of secreted proteins:

• polypeptide hormones (e.g., insulin)

• albumin

• collagen

• immunoglobulins

• Integral membrane proteins are also synthesized by the same mechanisms;
they may be considered “partially secreted”

• Examples of integral membrane proteins:

• polypeptide hormone receptors (e.g., insulin receptor)

• transport proteins

• ion channels

• cytoskeletal anchoring proteins (e.g., band 3)

Some examples of posttranslational modications.

Org. Biomol. Chem.2, 1, 2004

Classical protein translation and mechanisms of control. (a) Major events

in translation-initiation are: (i) the

eIF4F initiation complex, consisting of

eIF4A, eIF4G, and eIF4E

to the m7GpppN cap, is recruited and is joined by

eIF4B; (ii) the

48S ribosomal initiation complex

is formed by the joining of

the 43S pre-initiation complex, which contains eIF3, the 40S ribosomal

subunit and eIF2-GTP-Met-tRNAi, (iii) the 48S complex scans across the 5’-

UTR to the AUG initiation codon; and (iv) AUG is recognized by the

anticodon in Met-tRNAi and the 60S ribosomal subunit is recruited, which

is accompanied by hydrolysis of eIF2-bound GTP and dissociation of

initiation factors, to form a translation-competent 80S ribosome. during

protein elongation (v-vii), multiple 80S ribosomes simultaneously transit

the open reading frame (ORF) while elongating the newly synthesized

peptide chain [which requires aminoacid-charged tRNAs and elongation

factors (notshown)]. During termination (viii), the ribosome recognizes a

stop codon and the 80S ribosomal subunits dissociate from the mRNA in a

process requiring release factors (not shown). Also shown are

5’- and 3’-

untranslated region (UTR)-binding proteins

.

TIBS 28, 91, 2003

Initiation of protein synthesis in eukaryotesis important both
mechanically, because it selects the translation reading frame, and
biologically, because It is the primary site for regulation of
translation. For approximately 95–98% of the cellular mRNAs,
formation of an initiation complex follows a rather specific pathway
that is broken down in to seven discrete steps.

Although almost 35

peptids in12–14 translation initiation factors participate in this
process, three appear to have dominant roles: eukaryotic initiation
factor (

eIF

)

3

, in building a pool of 40S subunits;

eIF2

, in binding the

initiator tRNA (tRNAi) to the 40S subunit; and

eIF4F

inactivating the

mRNA and binding it to the 40S subunit.

This process requires both

ATP and GTP. There sulting 80S initiation complex contains both the
tRNAi and them RNA, with the anticodon of the tRNAi correctly base
paired with the initiating AUG code word. Regulation of translation
focuses mostly on controlling the activity of either eIF2 or eIF4F and
this regulation has different consequences. Reduction in eIF2 activity
influences all mRNA approximately the same, where as reductionin
eIF4F activity drives competition between mRNAs.

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION 31, 378, 2003

Initiation of Protein Biosynthesis in Eukaryotes