GTP-binding proteins

Molecular Biochemistry II

Translation: Protein Synthesis

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/translate.htm

Contents of this page:
GTP-binding proteins
Initiation of protein synthesis
Elongation
Termination
Eukaryotic translation

Introduction: Coverage of this topic will be limited. Essential details of protein synthesis are covered in many courses, and are presented well in the textbook. These notes will focus on structural aspects, and on protein factors involved in initiation, elongation, and termination of protein synthesis, many of which are GTP-binding proteins, and 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.

GTP-binding proteins

Heterotrimeric G-proteins, and the related family of small GTP-binding proteins, are 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 + P_i converts the protein to the inactive conformation. Reactivation occurs by release of bound GDP in exchange for GTP.

Small GTP-binding proteins require helper proteins to facilitate GDP/GTP exchange or to promote GTP hydrolysis.

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 is usually present at higher concentration than GDP. Thus a GEF causes a GTP-binding protein to release GDP and bind GTP (GDP/GTP exchange). A GTPase activating protein (GAP) causes a GTP-binding protein to hydrolyze its bound GTP to GDP + Pi. The active site for GTP hydrolysis is on the GTP-binding protein, although the GAP may contribute an essential active site residue.

GEFs and GAPs may be separately regulated. Unique GEFs and GAPs interact with different GTP-binding proteins.

Members of the family of small GTP-binding proteins have diverse functions. In some cases, the difference in conformation associated 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.F o r a list of some small GTP-binding proteins and their roles, see the section on cell signals.

Initiation of protein synthesis in E. coli requires initiation factors IF-1, IF-2, and IF-3. The sequence of events is summarized in the diagram on p. 1323 of Biochemistry, 3rd Edition, by Voet & Voet..

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. Binding of IF-1 also occludes the A site domain of the small ribosomal subunit, helping to insure that the initiation aminoacyl-tRNA, fMet-tRNA^fMet, can bind only in the P site and that no other aminoacyl-tRNA can bind in the A site during initiation.

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

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

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

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 (see Chime exercise below).

Elongation requires participation of elongation factors EF-Tu (also called EF1A), EF-Ts (EF1B) and EF-G (EF2). Two of these, EF-Tu and EF-G, are small GTP-binding proteins.

Elongation cycle: The diagram below, showing the positions of EF-Tu, EF-G, and tRNAs relative to the ribosome during the elongation cycle, was provided by Dr. Joachim Frank.

Colors: The large ribosome subunit is cyan, the small ribosome subunit pale yellow, EF-Tu red, and EF-G blue. tRNAs are gray (free or complexed with EF-Tu), magenta (binding at A site), green (in P site), yellow or brown (in the process of exiting).
Dr. Frank's laboratory group at the Wadsworth Center, New York State Department of Health, used cryo-EM and 3D image reconstruction to determine ribosomal structures and positions of EF-Tu and EF-G. Structures of EF-Tu and EF-G are based on separate X-ray crystallographic studies.

The sequence of events, as summarized in the diagram above and on p. 1327, is as follows:

EF-Tu -GTP binds and delivers an aminoacyl-tRNA to the A site on the ribosome. EF-Tu recognizes & binds all aminoacyl-tRNAs with approximately the same affinity, when each tRNA is bonded to the correct (cognate) amino acid. tRNAs for the different amino acids have evolved to differ slightly in structure, to compensate for different binding affinities of amino acid side-chains, so that the aminoacyl-tRNAs all have similar affinity for EF-Tu.

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. The change in ribosomal conformation 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.

 

When EF-Tu delivers the 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 and dissociates from the complex. The tRNA conformation relaxes, and the acceptor stem is repositioned to promote peptide bond formation. This process is called accommodation. Accommodation 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, and adjacent to the mouth of the tunnel through which nascent polypeptides exit the ribosome. The diagram at right is from the file to be explored by Chime below. For images depicting the proposed rotational movement, see Fig. 5B in a website maintained by A. E. Yonath.

EF-Ts functions as GEF to reactivate EF-Tu. Interaction with EF-Ts causes EF-Tu to release its bound GDP. Upon dissociation of EF-Ts, EF-Tu binds GTP, which is present in the cytosol at higher concentration than GDP.

The difference in conformation of EF-Tu, depending on whether GDP or GTP occupies its nucleotide binding site, is apparent from crystal structures to be viewed below. In two of the crystals, GDPNP, a non-hydrolyzable analog of GTP, is present in the nucleotide-binding site of EF-Tu.

Studio exercise: Students should 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 in the instructions, so that the images can be compared. Each student should view and compare all 3 structures by observing displays prepared by other members of the group:

1. EF-Tu with bound GDP
2. EF-Tu with the GTP analog GDPNP
3. EF-Tu with the GTP analog GDPNP and Phe-tRNA^Phe.

EF-Tu-GDP

EF-Tu-GDPNP

EF-Tu-GDPNP-aa-tRNA

Question: Does substitution of GTP (GDPNP) for GDP, or the binding of aa-tRNA, have a greater effect on conformation of EF-Tu?

Transpeptidation(peptide bond formation) involves nucleophilic attack of the amino N of the amino acid that is linked to the 3' hydroxyl 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. (Review Chime exercise on the large ribosomal subunit.) The 23S rRNA may be considered a "ribozyme." As part of the reaction a proton (H+) is extracted from the attacking amino N. This H+ is then donated to the hydroxyl of the tRNA in the P site as the ester linkage is cleaved. It had been proposed that a ring N of a highly conserved adenosine at the active site might 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. H+ shuttling is attributed instead to the adjacent ribose 2' hydroxyl group of the P-site peptidyl-tRNA, along with ribose hydroxyls of active site rRNA residues and 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.

 

The nascent polypeptide (one amino acid longer) is now linked to the A-site tRNA. However, translocation has already partly occurred, because of the earlier rotation of the single-stranded 3' end of the A-site tRNA toward the P-site, which positioned the aminoacyl moiety for catalysis. The unloaded tRNA in the P site will shift to the E (exit) site during translocation.

 

Translocation of the ribosome relative to the mRNA involves the GTP-binding protein EF-G. As depicted above in the diagram of the elongation cycle, the size and shape of EF-G are comparable to that of the complex of EF-Tu with an aa-tRNA. Structural studies and molecular dynamics simulations 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. The tRNA with attached nascent polypeptide is pushed from the A site to the P site, while the now unloaded tRNA that was in the P site shifts to the E site. Since the tRNAs are linked to the mRNA by codon-anticodon base pairing, the mRNA moves relative to the ribosome.

Colors: The large ribosome subunit is cyan, the small ribosome subunit pale yellow, EF-G blue and tRNAs green or yellow. mRNA is represented as a string of beads. This figure was provided by Dr. Joachim Frank of the Wadsworth Center, New York State Department of Health.

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

Interaction with the ribosome, which functions as GAP (GTPase activating protein) for EF-G, causes EF-G to hydrolyze its bound GTP to GDP + P_i. EF-G-GDP then dissociates from the ribosome. A domain of EF-G appears to function as its own GEF (guanine nucleotide exchange factor) to regenerate EF-G-GTP.

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 below the 30S moiety of a bacterial ribosome, complexed with a short genetically engineered mRNA, and with tRNA^Phe in each of the A, P, and E (exit) sites. Due to limited resolution, proteins and rRNA display only as backbone. File PDB-1GIX: Structure solved by M. M. Yusupov, G. Z. Yusupova, A. Baucom, K. Lieberman, T. N. Earnest, J. H. D. Cate & H. F. Noller in 2001. Structural information for the co-crystallized 50S subunit is in a separate data file (1GIY).

Recommended display options: Separately select and display each of the following as sticks with color CPK: chain b (tRNA in A-site) chain c (tRNA in P-site) Now select chain 1 (one) and display as ball & stick with color CPK. This is a small fragment of mRNA. Drag and zoom in to view codon/anticodon base-pairing between mRNA and tRNAs in the A and P sites. Note the proximity of acceptor stems of A-site & P-site tRNAs. Now select chain d (tRNA in E-site), display as sticks with color CPK. Select protein, display as backbone with color chain. Select chain a (16S rRNA), and display backbone. Question: Which constituents of the 30S ribosomal subunit predominantly interact with mRNA and tRNAs bound to A & P sites: rRNA or protein?

CO N S P

Chain termination requires participation of release factors RF-1, RF-2, and RF-3. RF-3 is a small GTP-binding protein. The process is summarized on p. 1335.

RF-1 and RF-2 recognize and 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 the release factors occupy the A site on the ribosome, the ribosomal Peptidyl Transferase catalyzes transfer of the peptidyl group to water (hydrolysis). Hydrolysis of GTP on RF-3, to GDP + P_i, 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.

Websites with animations depicting protein translation:

· 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, based on crystal structures of the ribosome and various protein factors.

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