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Protein Synthesis Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II
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Page 1: translation RNA

Protein Synthesis

Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved.

Molecular Biochemistry II

Page 2: translation RNA

The focus here will be on structural aspects, and on protein factors involved in initiation, elongation, & termination of protein synthesis.

Many of these factors are GTP-binding proteins, & other proteins that control GDP/GTP exchange or GTPase activity of these GTP-binding proteins.

Bacterial translation mechanisms will be emphasized.

The more complex process of mammalian translation and its regulation will be only briefly introduced.

Page 3: translation RNA

Heterotrimeric G-proteins, and the related family of small GTP-binding proteins, were introduced in the notes on cell signals.

A GTP-binding protein has a different conformation depending on whether it has bound to it GTP or GDP. 

Usually bound GTP stabilizes the active conformation.

Hydrolysis of the bound GTP to GDP + Pi converts

the protein to the inactive conformation.

Reactivation occurs by release of bound GDP in exchange for GTP.

Page 4: translation RNA

A guanine nucleotide exchange factor (GEF) induces a conformational change that makes the nucleotide-binding site of a GTP-binding protein more accessible to the aqueous intracellular milieu, where [GTP] [GDP].

Thus a GEF causes a GTP-binding protein to release GDP & bind GTP (GDP/GTP exchange).

G protein-GTP (active)

GDP

GEF GAP

GTP Pi

G protein-GDP (inactive)

Small GTP-binding proteins require helper proteins, to • facilitate

GDP/GTP exchange, or

• promote GTP hydrolysis.

Page 5: translation RNA

G protein-GTP (active)

GDP

GEF GAP

GTP Pi

G protein-GDP (inactive)

The active site for GTP hydrolysis is on the GTP-binding protein, although a GAP may contribute an essential active site residue.

GEFs & GAPs may be separately regulated.

Unique GEFs and GAPs interact with different GTP-binding proteins

A GTPase activating protein (GAP) causes a GTP-binding protein to hydrolyze its bound GTP to GDP + Pi.

Page 6: translation RNA

Members of the family of small GTP-binding proteins have diverse functions.

In some cases, the difference in conformation, with substitution of GDP for GTP allows a GTP-binding protein to serve as a "switch".

In other cases the conformational change may serve a mechanical role or alter the ability of the protein to bind to membranes.

Page 7: translation RNA

IF-3 binds to the 30S ribosomal subunit, freeing it from its complex with the 50S subunit.

IF-1 assists binding of IF-3 to the 30S ribosomal subunit. IF-1 also occludes the A site of the small ribosomal subunit, helping insure that the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation.

IF-2 is a small GTP-binding protein. IF-2-GTP binds the initiator fMet-tRNAfMet & helps it to dock with the small ribosome subunit.

Initiation of protein synthesis in E. coli requires initiation factors IF-1, IF-2, & IF-3.

Page 8: translation RNA

Once the two ribosomal subunits come together, the mRNA is threaded through a curved channel that wraps around the "neck" region of the small subunit.

As mRNA binds, IF-3 helps to correctly position the complex such that the tRNAfMet interacts via base pairing with the mRNA initiation codon (AUG). A region of mRNA upstream of the initiation codon, the Shine-Dalgarno sequence, base pairs with the 3' end of the 16S rRNA. This positions the 30S ribosomal subunit in relation to the initiation codon.

As the large ribosomal subunit joins the complex, GTP on IF-2 is hydrolyzed, leading to dissociation of IF-2-GDP and dissociation of IF-1. A domain of the large ribosomal subunit serves as GAP (GTPase activating protein) for IF-2.

Page 9: translation RNA

Colors: large ribosome subunit, cyan; small subunit, pale yellow; EF-Tu, red; EF-G, blue. tRNAs, gray, magenta, green, yellow, brown.

Elongation cycleRibosome structure and position of factors & tRNAs based on cryo-EM with 3D image reconstruction. Diagram provided by Dr. J. Frank, Wadsworth Center, NYS Dept. of Health.Partial images on subsequent slides are derived from this.

Page 10: translation RNA

Elongation requires participation of elongation factors• EF-Tu (also called EF1A)• EF-Ts (EF1B)• EF-G (EF2)

EF-Tu & EF-G are small GTP-binding proteins.

The sequence of events follows.

Page 11: translation RNA

EF-Tu-GTP binds & delivers an aminoacyl-tRNA to the A site on the ribosome.

EF-Tu recognizes & binds all aminoacyl-tRNAs with approx. the same affinity, when each tRNA is bonded to the correct (cognate) amino acid.

tRNAs for different amino acids have evolved to differ slightly in structure, to compensate for different binding affinities of amino acid side-chains, so the aminoacyl-tRNAs all have similar affinity for EF-Tu.

EF-Tu colored red

Page 12: translation RNA

The tRNA must have the correct anticodon to interact with the mRNA codon positioned at the A site to form a base pair of appropriate geometry.

Universally conserved bases of 16S rRNA interact with and sense the configuration of the minor groove of the short stretch of double helix formed from the first 2 base pairs of the codon/anticodon complex.

A particular ribosomal conformation is stabilized by this interaction, providing a mechanism for detecting whether the correct tRNA has bound.

Proofreading in part involves release of the aminoacyl-tRNA prior to peptide bond formation, if the appropriate ribosomal conformation is not generated by this interaction.

Page 13: translation RNA

The change in ribosomal conformational associated with formation of a correct codon-anticodon complex leads to altered positions of active site residues in the bound EF-Tu, with activation of EF-Tu GTPase activity.

The ribosome thus functions as GAP for EF-Tu.

EF-Tu-GTP ribosome (GAP) Pi EF-Tu-GDP

Page 14: translation RNA

When EF-Tu delivers an aminoacyl-tRNA to the ribosome, the tRNA initially has a distorted conformation.

As GTP on EF-Tu is hydrolyzed to GDP + Pi , EF-Tu undergoes a large conformational change & dissociates from the complex.

The tRNA conformation relaxes, & the acceptor stem is repositioned to promote peptide bond formation.

This process is called accommodation.

EF-Tu colored red

Page 15: translation RNA

It includes rotation of the single-stranded 3' end of the acceptor stem of the A-site tRNA around an axis that bisects the peptidyl transferase center of the ribosomal large subunit.

This positions the 3' end with its attached amino acid in the active site, near the 3' end of the P-site tRNA, & adjacent to the mouth of the tunnel through which nascent poly-peptides exit the ribosome.

acceptor stems of P-site & A-site tRNAs

PDB 1GIX

For images depicting the proposed rotational movement, see Fig. 5B in website of A. E. Yonath.

Page 16: translation RNA

EF-Tu-GTP*

GDP

EF-Ts (GEF) ribosome (GAP)

GTP Pi

EF-Tu-GDP**

*EF-Tu-GTP (conformation 1) binds &delivers aa-tRNA to A site on ribosome.

**EF-Tu-GDP (conformation 2)dissociates from complex.

Interaction with EF-Ts causes EF-Tu to release GDP.

Upon dissociation of EF-Ts, EF-Tu binds GTP, which is present in the cytosol at higher concentration than GDP.

EF-Ts functions as GEF to reactivate EF-Tu.

Page 17: translation RNA

The difference in conformation of EF-Tu, depending on whether GDP or GTP occupies its nucleotide binding site, is apparent in crystal structures to be viewed by Chime.

In 2 of the crystals, GDPNP, a non-hydrolyzable analog of GTP, is present in the nucleotide-binding site of EF-Tu.

NH

N

N

O

NH2N

O

OHOH

HH

H

CH2

H

OPOPNPO

O

O

O

O

O

O

H

GDPNP

Page 18: translation RNA

Compare, using Chime, the structures of: EF-Tu with bound GDP EF-Tu with bound GTP analog GDPNP EF-Tu with bound GTP analog & Phe-tRNAPhe

Work in groups of 3, with one of the 3 files assigned to each student in the group.

Please use colors and displays exactly as specified, so that images can be compared.

Each student should examine all 3 structures by observing displays prepared by other group members.

Question: Does substitution of GTP (GDPNP) for GDP, or binding of aa-tRNA, affect EF-Tu conformation more?

Page 19: translation RNA

Transpeptidation (peptide bond formation) involves nucleophilic attack of the amino N of the amino acid linked to the 2' or 3' OH of the terminal adenosine of the tRNA in the A site on the carbonyl C of the amino acid (with attached nascent polypeptide) in ester linkage to the tRNA in the P site.

The reaction is promoted by the geometry of the active site consisting solely of residues of the 23S rRNA of the large ribosomal subunit.  No protein is found at the active site.

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH2

R

C

HC

NH3+

R

O

O

O

:

P site A site

Page 20: translation RNA

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH2

R

C

HC

NH3+

R

O

O

O

:

P site A site

The 23S rRNA may be considered a "ribozyme." 

As part of the reaction a proton (H+) is extracted from the attacking amino N.

Page 21: translation RNA

This H+ is then donated to the hydroxyl of the tRNA in the P site, as the ester linkage is cleaved.

O

OHOH

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

C

HC

NH

R

C

HC

NH3+

R

O

O

P site A site

Page 22: translation RNA

It has been proposed that a ring N of a highly conserved adenosine at the active site may act as a catalyst mediating this H+ transfer.

However, on the basis of recent structural and mutational evidence it has been concluded that the active site adenine is essential only as part of the structure of the active site that positions the substrates correctly.

Proton shuttling is attributed instead to the adjacent ribose hydroxyl of the P-site substrate, along with ribose hydroxyls of active site rRNA residues & structured water molecules that collectively form a H-bonded network at the active site.

For a diagram see Fig 5 of the review by Rodnina et al.

Page 23: translation RNA

The nascent polypeptide, one residue longer, is now linked to the A-site tRNA.

O

OHOH

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

C

HC

NH

R

C

HC

NH3+

R

O

O

P site A site

Page 24: translation RNA

However, translocation has already partly occurred, because peptide bond formation is associated with rotation of the single-stranded 3' end of the A-site tRNA toward the P-site, positioning the aminoacyl moiety for catalysis.

This rotary movement also positions the nascent polypeptide to feed into the entrance to the protein exit tunnel, which is located midway between A & P sites.

Page 25: translation RNA

The unloaded tRNA in the P site will shift to the E (exit) site during translocation.

Translocation of the ribosome relative to mRNA involves the GTP-binding protein EF-G.

The size & shape of EF-G are comparable to that of the complex of EF-Tu with an aa-tRNA.

Structural studies & molecular dynamics indicate that EF-G-GTP binding in the vicinity of the A site causes a ratchet-like motion of the small ribosomal subunit against the large subunit.

tRNA grey, EF-Tu red, EF-G blue

Page 26: translation RNA

The tRNA with attached nascent polypeptide is pushed from the A site to the P site.

Unloaded tRNA that was in the P site shifts to the E site.

Since tRNAs are linked to mRNA by codon-anticodon base pairing, the mRNA moves relative to the ribosome.

small subunit

large subunit

mRNAlocation

EF-G

tRNA

Figure provided by Dr. J. Frank, Wadsworth Center.

Page 27: translation RNA

Additionally, it has been postulated that translocation is spontaneous after peptide bond formation because:

• the deacylated tRNA in the P site has a higher affinity for the E site, &

• the peptidyl-tRNA in the A site has a higher affinity for the P site.

Interaction with the ribosome, which acts as GAP (GTPase activating protein) for EF-G, causes EF-G to hydrolyze its bound GTP to GDP + Pi.

EF-G-GDP then dissociates from the ribosome.

A domain of EF-G functions as its own GEF (guanine nucleotide exchange factor) to regenerate EF-G-GTP.

Page 28: translation RNA

The continued codon-anticodon base paring of the tRNA in the E site is postulated to have a role in preventing potentially serious frame-shift errors, e.g., such as would occur if the tRNAs were to able to shift laterally by one base pair.

Normally the empty tRNA is released from the E site only after binding of the correct aminoacyl-tRNA at the A site causes a decreased affinity for tRNA in the E site

Explore with Chime the 30S moiety of a bacterial ribosome, complexed with a short, genetically engineered mRNA, and with tRNAPhe in the A, P, & E sites.

Page 29: translation RNA

Chain termination requires release factors RF-1, RF-2, & RF-3. RF-3 is a small GTP-binding protein.

RF-1 & RF-2 recognize & bind to STOP codons. One or the other binds when a stop codon is reached.

RF-3-GTP facilitates binding of RF-1 or RF-2 to the ribosome.

Page 30: translation RNA

Once release factors occupy the A site, Peptidyl Transferase catalyzes transfer of the peptidyl group to water (hydrolysis).

Hydrolysis of GTP on RF-3 causes a conformational change that results in dissociation of release factors.

A ribosomal recycling factor (RRF) is required, with EF-G-GTP and IF-3, for release of uncharged tRNA from the P site, and dissociation of the ribosome from mRNA with separation of the two ribosomal subunits.

Page 31: translation RNA

Websites with animations:

Animation of protein elongation from the laboratory of J. Frank of the Wadsworth Center, based on Cryo-EM and X-Ray observations of structures of the ribosome, elongation factors, and tRNA.

Animation of the ribosome in translation from the laboratory of V. Ramakrishnan of the MRC Laboratory of Molecular Biology, based on crystal structures of the ribosome and various protein factors.

Page 32: translation RNA

Eukaryotic Translation

Translation of mRNA is highly regulated in multi-cellular eukaryotic organisms, whereas in prokaryotes regulation occurs mainly at the level of transcription.

There is global regulation of protein synthesis.

E.g., protein synthesis may be regulated in relation to the cell cycle or in response to cellular stresses such as starvation or accumulation of unfolded proteins in the endoplasmic reticulum.

Mechanisms include regulation by signal-activated phosphorylation or dephosphorylation of initiation and elongation factors.

Page 33: translation RNA

Translation of particular mRNAs may be inhibited by small single-stranded microRNA molecules about 20-22 nucleotides long.

MicroRNAs bind via base-pairing to 3' un-translated regions of mRNA along with a protein complex RISC (RNA-induced silencing complex), inhibiting translation and in some cases promoting mRNA degradation.

Tissue-specific expression of particular genome-encoded microRNAs is an essential regulatory mechanism controlling embryonic development.

Some forms of cancer are associated with altered expression of microRNAs that regulate synthesis of proteins relevant to cell cycle progression or apoptosis.

Page 34: translation RNA

Protein factors that mediate & control translation are more numerous in eukaryotes than in prokaryotes.

Eukaryotic factors are designated with the prefix "e".

Some factors are highly conserved across kingdoms.

E.g., the eukaryotic elongation factor eEF1A is structurally and functionally similar to the prokaryotic EF-TU (EF1A).

In contrast, eEF1B, the eukaryotic equivalent of the GEF EF-Ts, is relatively complex, having multiple subunits subject to regulatory phosphorylation.

Page 35: translation RNA

Initiation of protein synthesis is much more complex in eukaryotes, & requires a large number of protein factors.

Some eukaryotic initiation factors (e.g., eIF3 & eIF4G) serve as scaffolds, with multiple domains that bind other proteins during assembly of large initiation complexes.

Page 36: translation RNA

Usually a pre-initiation complex forms, including: several initiation factors the small ribosomal subunit the loaded initiator tRNA, Met-tRNAi

Met.

This then binds to a separate complex that includes: mRNA initiation factors including ones that interact with

the 5' methylguanosine cap & the 3' poly-A tail, structures unique to eukaryotic mRNA.

Within this complex mRNA is thought to circularize via interactions between factors that associate with the 5' cap & with a poly-A binding protein. A simplified diagram of the eukaryotic initiation complex once it has reached the initiation codon is found in the WormBook.

Page 37: translation RNA

After the initiation complex assembles, it translocates along the mRNA in a process called scanning, until the initiation codon is reached.

Scanning is facilitated by eukaryotic initiation factor eIF4A, which functions as an ATP-dependent helicase to unwind mRNA secondary structure while releasing bound proteins.

A short sequence of bases adjacent to the AUG initiation codon may aid in recognition of the start site.

After the initiation codon is recognized, there is hydrolysis of GTP and release of initiation factors, as the large ribosomal subunit joins the complex and elongation commences.

Page 38: translation RNA

See Fig. 1 of the article by Hinnebusch (requires TIBS subscription).

Some eukaryotic mRNAs have what is called an internal ribosome entry site (IRES), far from the 5' capped end, at which initiation may occur without the scanning process.