• There are many steps to produce a protein in a eukaryotic cell

• Each step is a point of regulation to determine the efficiency of gene expression

Translation

• Initiation

• Elongation

• Termination

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.

Heterotrimeric G-proteins.

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

Usually GTP binding induces 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.

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.

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.

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.

Roles of some small GTP-binding proteins:

IF-2, EF-Tu, EF-G, & RF-3: Protein synthesis initiation, elongation, & release factors.

Ras: Growth factor signal cascades.

Rab: Membrane vesicle targeting & fusion.

ARF: Vesicle budding by formation of coatomer coats.

Ran: Transport of proteins into & out of the nucleus.

Rho: Regulation of the actin cytoskeleton.

Translation initiation

Eukaryotes v. Prokaryotes

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 tRNAfMet will end up 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 tRNAfMet & helps it to dock with the small ribosome subunit.

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

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 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, helping to position the 30S ribosomal subunit in relation to the initiation codon.

The large ribosomal subunit then 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.

Initiation

Shine Delgarno Sites

initiation of translation in eukaryotes

1. charging of the tRNAs is the same

2. Association of the translation machinery- terminology difference initiation factors called eIFs rather than IFs- charged tRNA is delivered by eIF2-GTP (which is hydrolized to GDP to provide energy)- eukaryotic mRNA is capped, this is recognized by an additional protein eIF-4E

3. identification of initiator codon- ribosome tRNA complex scans for first AUG and stops there- directed by the eIF-4E on the CAP site rather than the Shine-Delgarno site

4. completion of initiation- same

Translation Initiation - Eukaryotes

Controlling Initiation

• Eukaryotic mRNA has cap on 5’ end• Bacteria have no “5’ end marker”

– Translation is coupled to transcription– Ribosomes bind to RNA as it is made

• Where to start reading mRNA?– Shine Delgarno sequence in bacteria, Kozak

sequence in eukaryotes– Adjacent to the first codon

The AUG start codon is recognized by methionyl-tRNAi

Met

Translation elongation

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.

Elongation requires participation of elongation factors• EF-Tu (also called EF-1A)• EF-Ts (EF-1B)• EF-G (EF-2)

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

The sequence of events follows.

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

The loaded tRNA must have the correct anticodon to base pair with the mRNA codon positioned at the A site.

tRNA binding causes a conformational change in the small ribosomal subunit that causes universally conserved bases of 16S rRNA to interact closely with the minor groove of the first two base pairs of the codon/anticodon complex, helping insure that only the correct tRNA binds.

Proofreading in part involves release of the aa-tRNA prior to peptide bond formation if a particular ribosomal conformation is not stabilized by this interaction.

As the aa-tRNA is delivered by EF-Tu to the A site on the ribosome, GTP on EF-Tu is hydrolyzed to GDP + Pi.

A domain of the ribosome serves as GAP for EF-Tu.

This function depends on codon-anticodon recognition correctly positioning the aa-tRNA in relation to the large ribosomal subunit. EF-Tu colored red

A large conformational change in EF-Tu, when GTP GDP + Pi, promotes dissociation of EF-Tu.

Release of EF-Tu leads to repositioning of the aa-tRNA to promote peptide bond formation.

EF-Tu colored red

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.

EF-Ts induces EF-Tu to release bound GDP & bind GTP.

EF-Ts dissociates from EF-Tu when EF-Tu changes its conformation, upon binding GTP.

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

Transpeptidation (peptide bond formation) involves acid/base catalysis by a universally conserved adenosine of the 23S rRNA of the large ribosomal subunit.

No protein is found adjacent to the active site adenosine. (Recall Chime exercise on the large ribosomal subunit.)

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

The amino N of the amino acid linked to the 2' or 3' OH of the terminal adenosine of tRNA in the A site reacts with the carbonyl C of the amino acid (with attached nascent polypeptide) linked to the tRNA in the P site.

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

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

However, peptide bond formation is associated with rotation of the acceptor stem of the A site tRNA, so that the nascent polypeptide is positioned to feed via the P site into

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

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.

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

EF-G-GTP binds in the vicinity of the A site.

EF-G-GTP binding may push the tRNA with attached nascent polypeptide from the A site to the P site.

Unloaded tRNA that was in the P site shifts to an exit site.

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

small subunit

large subunit

mRNAlocation

EF-G

tRNA

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.

Bacterial Elongation

• Elongation factor Tu uses GTP hydrolysis to help bring tRNA to A site

• Elongation factor Ts helps restore fresh GTP to EF-Tu

• Elongation factor G uses GTP hydrolysis to help move tRNA from A to P site

Eukaryotic Elongation

• One factor (eEF-1) does job of EF-Tu and EF-Ts

• eEF-2 does job of EF-G

• As with bacteria, the elongation factors work within the ribosome

Translation termination

Eukaryotes v. Prokaryotes

Termination

• Three codons lack complementary tRNAs• Recognized by “release factors”

– Three proteins in bacteria– Two can bind to “stop codon”– Third helps with interaction, uses GTP– Eukaryotes have one release factor

• Induce the breakage of tRNA-amino acid bond of tRNA in P site

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.

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

Hydrolysis of GTP on RF-3 causes a conformational change that results in dissociation of the release factors. The ribosome dissociates from mRNA.

IF-3, assisted by IF-1, promotes dissociation of the two ribosomal subunits for another round of initiation.

5’ 3’

5’ 3’

peptide chain-

GCCAUG

GCCAUG UAA

A

UUU

A: (aminoacyl) siteP: (peptidyl) siteE: (exit) site

A

termination, directed by the STOP codon

EF-G-GTPEF-G-GDP

Release factorpeptide chain-

peptide chain-

Polyribosomes. these structures are formed by the presence of several ribosomes working sequentially on a single mRNA

The ribosome by SEM

Functional concept of the ribosome

Antibiotic Action

Puromycin Mechanism

Cap-dependent vs.cap-independent translation initiation

Cap-Dependent Cap-Independent

43S particle recruitment strategies