Biochemistry

Chen Yonggang

Zhejiang University Schools of Medicine

Translation, making protein following nucleic acid directions

Bodega Bay, Sonoma County

Breakfast at The Tides, Bodega Bay

The process of using base pairing language to create a protein is termed

Translation• Any process requires:

– A mechanism Ribosome

– Information-directions mRNA

– Raw materials amino acids / tRNA

– Energy ATP

• Any process has stages:– Beginning Initiation

– Middle Elongation

– End Termination

Translation requires a Dictionary

• The dictionary of Translation is called the Genetic Code [Table 6.1]

• Correlates mRNA with Protein– 3 nucleotides = 1 amino acid 43= 64

• 4 possible nts 20 possible aa

• 3 nucleotides read 5’→3’ are called a codon

– Codes for 1 amino acid

The Genetic Code

The Genetic Code

• Triplet made of codons• Non-overlapping read sequentially• Unpunctuated once started, set frame• Degenerate > than one codon/AA• Nearly universal mitochondrial code• Start signals AUG[met]• Stop signals UAG, UAA, UGA

Players in Translation

• Ribosome the machinery

• mRNA the information

• Aminoacyl-tRNA the translator!– Amino Acids/tRNA– ATP

Ribosomes are ribonucleoprotein complexes table 6.7

Small subunit

Large Subunit

PROCARYOTIC EUCARYOTIC

70 S

30S

50S

RNA 5S, 16S, 23S

PROTEINS 55

80 S

40S

60S

RNA 5S, 5.8S,18S,28S

PROTEINS 84

Ribosomes must be assembled with an mRNA

• The initiation process requires protein factors

• A mRNA must be recognized and reading frame must be set

• Aminoacyl-tRNAs must be available

5’ 3’

Since the Translator is the Aminoacyl-tRNA, it must be important

• Cells have 30+ tRNAs

• tRNAs are redundant for some amino acids

• Cells have 20 Aminoacyl-tRNA Synthetases

• Aminoacyl-tRNA synthetases recognize 1 amino acid and 1 or more tRNAs

• Aminoacylation is very precise

Aminoacyl-tRNA Synthetases are critical to Translation

• 1 Aminoacyl-tRNA Synthetase recognizes 1 Amino Acid and binds it

• 1 Aminoacyl-tRNA Synthetase recognizes 1 or more tRNAs specific for 1 amino acid

• The aminoacyl-tRNA Synthetase catalyzes a two step reaction which overall is

AAx + tRNAx + ATP AAx-tRNAx + AMP + PPiPage 239

The first step involves forming an enzyme-bound aminoacyl adenylate

ATP R CH

NH3

+CO

OA

OH

OH

OO CH2R CH

NH3

+CO

OPO

O+ +E

E..

+ PPi

The hydrolysis of the PPi makes the process irriversible

The second step transfers the amino acid to the 3’OH of the tRNA, retaining the

energy of the adenylate

A

OH

OH

OO CH2R CH

NH3

+CO

OPO

O

AMP

A

OH

OH

OO CH2

OPO

O-

+

tRNA

..

-

tRNAR CH

NH3

+

C OO

A

OH

OO CH2

OPO

O

tRNAs fold into L-shaped structuresFigure 2.59

Functional Sites of tRNAsFigure 2.58

• CCAOH 3’ Acceptor Sequence

• Amino acid acceptor stem

• D stem and loop

• Extra loop

• Anticodon stem and loop

• Anticodon

• TC stem and loop

• 5’ Terminus

The anticodon forms antiparallel base pairs with a codon in the mRNA

• Each tRNA has a unique anticodon

• There are 61 codons which base pair with tRNA anticodons, most pairing is Watson-Crick but Wobble in the 5’ base of the anticodon allows degeneracy

• 3 codons do not normally base pair with anticodons-UAA, UAG, UGA. The lack of a complementary anticodon-Termination Codons

Wobble allows one codon to base pair with up to three anticodons

Base stacking in the anticodon assures that bases 2 and 3 of the anticodon will follow Watson-Crick rules. Base 1 can wobble

GAG5' 3'

GCUmRNA

5'3'

cys

UGC

ACG

tRNA

Depending on base 1 it can pair with 1,2 or 3 bases

• If the wobble base is U, it can H bond to A (expected) or G (unexpected).

• If the wobble base is G, it can H bond to C (expected) or U (unexpected).

• A and C form only the expected base pairs.• Inosine in the wobble position can H bond

to A, C, and U.

Thus 31 tRNAs can read 61 codons

Translation takes place in three stages

• Initiation-- once per protein it gets the system in motion

• Elongation-- repeated for each codon in the mRNA making a peptide bond

• Termination-- finishes and releases the newly synthesized protein

Initiation

A common mechanism

Procaryotic initiation assembles the pre-translational complex

• Mechanism is similar for eucaryotes and procaryotes [differences are important]

• Components:– Small subunit containing a specific mRNA

sequence(Shine-Dalgarno) which guides the mRNA into correct position for reading frame relative to the 16S rRNA

– Proteinaceous initiation factors– Initiator AA-tRNA– mRNA(monocistronic for eucaryotes, polycistronic for

procaryotes)

Differences in the process provide the basis for specific antibiotic action

• Procaryotes

• 30S ribosomal subunit• IF-1, IF-2, IF-3• fMet-tRNAMetF

• GTP

• Eucaryotes• 40S ribosomal subunit• eIF-2a, eIF-3, eIF-4a, eIF-

4c, eIF-4e, eIF-4g, eIF-5, eIF-6

• Met-tRNAMeti

• GTP

Initiation Factors have Specific Roles

• Procaryotes

• IF-3 binds 30S• IF-2 binds initiator

AA-tRNA • IF-1 GTP hydrolysis• RNA:RNA base

pairing indexes mRNA

• Eucaryotes

• eIF-2 itRNA Binding

• eIF-3 40S anti-association

• eIF-4g binds mRNA

• eIF-4e cap binding

• eIF-4a mRNA indexing

• eIF-4c ribosomal i AA-tRNA

• eIF-5 GTP hydrolysis

• eIF-6 60S anti-association

In procaryotes IFs 1,2 and 3 are needed to begin

IF-3 is an 30S anti-association factor

IF-2 binds and preps initiator AA-tRNA

IF-1 is a GTP binding hydrolase

These allow the association of the 30S, Met-tRNA metF and factors to bind in preparation for mRNA and 50S binding

Initiation is similar for pro- and

eucaryotesDevlin 6.7

Intiation occurs once per translational cycle

• The preinitiation complex is formed on the small subunit

• GTP is bound to initiation factors. GTP hydrolysis carries out a process and drives a conformational change which leads to the next activity

• The mRNA is indexed to appropriate AUG codon• The mRNA is locked into the cleft between small and

large subunits• Addition of the large subunit creates A , P and E sites

on the ribosome• The initiator AA-tRNA is locked into the P site

Devlin 6.7

Eucaryotic initiation is similar

Eucaryotic initiation has differences

• The mRNA is not indexed by the ribosomal rRNA (eukaryotic mRNAs do not have Shine-Dalgarno sequence)

• Cap binding is essential for initiation• The initiation complex does not use formylated

methionine but does use a specific initiator Methionine-specific aminoacyl-tRNA for initiation

• Protein synthesis occurs at the first AUG

The association of all initiation components creates a 70S ribosome

with initiator tRNA in the P site

AUG

E P A

5' 3'CAU GCUmRNA

UAC

fmet

Elongation

A repeated experience

Once initiation is complete the ribosome is ready for elongation

• Elongation is the process of addition of amino acids to the C-terminus of the growing polypeptide

• Synthesis of each peptide bond requires energy derived from the cleavage of the AA-tRNA ester bond. The ribosomal enzyme doing this is called Peptidyl Transferase

• Elongation is repeated as many times as there are codons in the mRNA

As is the case for initiator tRNA all aminoacyl-RNAs must be present for

protein synthesis

• Good nutrition requires that all amino acids must be available in the diet

• For procaryotes most can be synthesized at an expense of energy

• Eucaryotes are able to form some but not all amino acids, thus some are essential in the diet

Pools of AA-tRNAs are formed by the Aminoacyl-tRNA Synthetases

• AA-tRNA synthetases recognize 2o and 3o structure near the TC,D, and extra loop and the acceptor stem on the L-shaped tRNA molecules

• AA-tRNA synthetases recognize 3-dimensional structure and functional groups of the amino acids

• As we saw earlier, AA-tRNA synthetases use ATP to form a high-energy ester bond at the 3’OH on the tRNA

Once an AAx-tRNAx is formed, the Amino Acid becomes Invisible

• The ribosome mediates the association between codons on the mRNA and anticodons on the tRNA

• Specificity of AA incorporation depends upon the anticodon of the tRNA

• Whatever is on the tRNA will be incorporated into the protein at the site

• The tRNA adapts the AA to the specified site

Following Initiation the Ribosome has 3 functional sites

• A site-aminoacyl-tRNA binding site [incoming AA-tRNA, only initiator AA-tRNA goes to the P site]

• P site-peptidyl-tRNA binding site[attachment of growing polypeptide site

• E site-spent tRNA exit site

APE

Each elongation cycle requires elongation factors

• Procaryotes

• EF-T AA-tRNA binding to A site, GTP binding/hydrolysis

• EF-G GTP hydrolysis, ribosomal conformational change, index peptidyl-tRNA to P site, expulsion of spent tRNA from E site

• Eucaryotes

• EF-1 AA-tRNA binding to A site, GTP binding/hydrolysis

• EF-2 GTP hydrolysis, ribosomal conformational change, index peptidyl-tRNA to P site, expulsion of tRNA from E site

In procaryotes, under the control of EF-T, a second aminoacyl-tRNA is bound in the A site

AUG

E P A

5' 3'CAU GCUmRNA

UAC

fmet

GUA

his

Devlin 6.8

In eucaryotes similar events occur

Hydrolysis of bound GTP changes the conformation of the Ribosome

• The conformational change locks the aminoacyl-tRNA into the A site

• Brings the anticodon in close approximation with the codon

• Prepares the ribosome for binding of another GTP binding hydrolase EF-G

The energy for peptide bond formation derives from the aminoacyl-tRNA ester bond

• Cleaving the ester bond provides energy for the formation of a peptide bond

• Catalysis is most likely provided by an integral 50/60S ribozyme, the peptidyl transferase, an RNA-containing enzyme(parts of the 23s rRNA) in the ribosome

• Upon synthesis of the peptide bond, the growing polypeptide chain is linked to the tRNA on the P site

Peptidyl transferase synthesizes a peptide bond forming a dipeptide

AUG

E P A

5' 3'CAU GCUmRNA

UAC

fmet

GUA

his

The peptide bond is formed using the energy derived from the aminoacyl ester bond and moves the peptide to the A site-bound Aminoacyl-tRNA

Following peptide bond formation a new factor drives translocation of the peptide

• Specificity provided by antiparallel codon-anticodon pairing between A site-bound AA-tRNA and mRNA

• Translocation driven by EF-G/2 catalyzed GTP hydrolysis-derived conformational change

• mRNA ratchets 5’→3’ through the ribosome moving the C(codon):AC(anticodon) from A to P site by the action of a translocase

• Time to find AA-tRNA is important to fidelity

EF-G mediated GTP hydrolysis translocates the mRNA and peptidyl-

tRNA expelling the spent tRNA

AUG

EP

A

5' 3'CAU GCUmRNA

UAC

fmet

GUA

his

Devlin 6.8

Eucaryotic translocation is similar

This elongation cycle is repeated as many times as there are codons

EF-T/1 mediated binding is followed peptide bond formation and EF-G/2

mediated peptidyl transfer

Eucaryotic elongation is similar to the procaryotic process

Repeat of 3 steps in elongation cycle

1. Binding of an incoming AA-tRNA

2. Peptide bond formation, catalyzed by

peptidyl transferase

3. translocation, done by translocase

The growing polypeptide chain remains attached to the last tRNA added

The next codon is UAG

When a termination codon occupies the the A site no AA-tRNA will bind

• Termination codons work because no tRNA has a complementary anticodon

• When the site is occupied by UAA, UAG or UGA time passes without A site occupancy by an AA-tRNA

• This allows binding of release or termination factors, proteins[size and shape of tRNAs] that change the activity of peptidyl transferase to a peptidyl hydrolase and thus mediate release of the polypeptide from the ribosome

Termination requires proteinaceous termination factors

• Procaryotes• Release Factor GTP

binding, GTP hydrolysis, conformational change, cleavage of 3’-peptidyl- CCAOH ester linkage, expulsion of polypeptide, dissociation of 30S and 50S subunits

• Eucaryotes• eRF GTP binding, GTP

hydrolysis, conformational change, cleavage of 3’-peptidyl-CCAOH ester linkage, expulsion of polypeptide, dissociation of 40S and 60S subunits

Devlin 6.10

Polysome

In both prokaryotes and eukaryotes, mRNAs are read simultaneously by numerous ribosomes, An mRNA with several ribosomes bound to it is referred to as a polysome.

Posttranslational modification

• Some newly made proteins, both prokaryotic and eukaryotic, do not attain their final biologically active conformation until they have been altered by one or more processing reactions called posttranslational modification

Different ways of modification

• Amino-Terminal and Carboxyl-Terminal Modification• Loss of Signal Sequence: the 15 to 30 residues at the

amino-terminal end of some proteins play a role in directing the protein to its ultimate destination in the cell. Such signal sequences are ultimately removed by peptidase

• Modification of Individual Amino Acids: The hydroxyl groups of Ser, Thr, and Tyr can be

phosphorylated , some others can be carboxylated and methylated.

Different ways of modification

• Attachment of Carbohydrate Side Chains: such as glycoproteins, N-linked oligosaccharides (e.g. Asn), O-linked-oligosaccharides(e.g. Ser or Thr)

• Addition of Isoprenyl Groups• Addition of Prosthetic Groups:Two examples are

the biotin molecule of acetyl-CoA carboxylase and the heme group of hemoglobin or cytochrome c.

Different ways of modification

• Proteolytic Processing: proinsulin and proteases such as chymotrypsinogen and trypsinogen(zymogen activation)

• Formation of Disulfide Cross-link: intrachain or interchain disulfide bridges between Cys residues

Because of differences in translation bacterial growth can be inhibited by

antibiotics

Devlin 6.8

Eucaryotes can be targeted by microorganisms

• Diphtheria toxin carries out its effects by mediating a covalent modification of eEF-2

NAD++ EF-2 ADP-Ribose-EF2 + Nicotinamide

• ADP-ribosylated eEF-2 is ineffective, thus interrupting polypeptide synthesis

What’s Next?

• Once made can proteins be modified?

• How is protein folding effected?

• How are proteins exported after synthesis?

• How is protein turnover controlled?