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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.

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biochemistry

Biochemistry

Chen Yonggang

Zhejiang University Schools of Medicine

the process of using base pairing language to create a protein is termed translation
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
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 code1
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
Players in Translation
  • Ribosome the machinery
  • mRNA the information
  • Aminoacyl-tRNA the translator!
    • Amino Acids/tRNA
    • ATP
ribosomes are ribonucleoprotein complexes table 6 7
Ribosomes are ribonucleoprotein complexes table 6.7

PROCARYOTIC

EUCARYOTIC

80 S

40S

60S

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

PROTEINS 84

70 S

30S

50S

RNA 5S, 16S, 23S

PROTEINS 55

Small subunit

Large Subunit

ribosomes must be assembled with an mrna
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

3’

5’

since the translator is the aminoacyl trna it must be important
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
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 + PPi

Page 239

the first step involves forming an enzyme bound aminoacyl adenylate
The first step involves forming an enzyme-bound aminoacyl adenylate

The hydrolysis of the PPi makes the process irriversible

slide15
The second step transfers the amino acid to the 3’OH of the tRNA, retaining the energy of the adenylate
functional sites of trnas figure 2 58
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
  • TyC stem and loop
  • 5’ Terminus
the anticodon forms antiparallel base pairs with a codon in the mrna
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
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

depending on base 1 it can pair with 1 2 or 3 bases
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.
translation takes place in three stages
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

Initiation

A common mechanism

procaryotic initiation assembles the pre translational complex
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

Differences in the process provide the basis for specific antibiotic action
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

Initiation Factors have Specific Roles
in procaryotes ifs 1 2 and 3 are needed to begin
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

intiation occurs once per translational cycle
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
eucaryotic initiation has differences
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
slide33

The association of all initiation components creates a 70S ribosome with initiator tRNA in the P site

elongation

Elongation

A repeated experience

once initiation is complete the ribosome is ready for elongation
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
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
Pools of AA-tRNAs are formed by the Aminoacyl-tRNA Synthetases
  • AA-tRNA synthetases recognize 2o and 3o structure near the TyC,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 aa x trna x is formed the amino acid becomes invisible
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
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

E

P

A

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

Each elongation cycle requires elongation factors
in procaryotes under the control of ef t a second aminoacyl trna is bound in the a site
In procaryotes, under the control of EF-T, a second aminoacyl-tRNA is bound in the A site
hydrolysis of bound gtp changes the conformation of the ribosome
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
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
slide46

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
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
EF-G mediated GTP hydrolysis translocates the mRNA and peptidyl-tRNA expelling the spent tRNA
repeat of 3 steps in elongation cycle
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

when a termination codon occupies the the a site no aa trna will bind
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

Termination requires proteinaceous termination factors
polysome
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
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
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 modification1
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 modification2
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
eucaryotes can be targeted by microorganisms
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
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?

I can hardly wait!