Cell and Molecular Biology Protein Synthesis Behrouz Mahmoudi
Cell and Molecular Biology Protein Synthesis Behrouz Mahmoudi www. soran. edu. iq 1
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. www. soran. edu. iq 2
Heterotrimeric G-proteins, and the related family of small GTP-binding proteins, were introduced in the notes on cell signals. w A GTP-binding protein has a different conformation depending on whether it has bound to it GTP or GDP. w Usually bound GTP stabilizes the active conformation. w Hydrolysis of the bound GTP to GDP + Pi converts the protein to the inactive conformation. w Reactivation occurs by release of bound GDP in exchange for GTP. www. soran. edu. iq 3
Small GTP-binding proteins require helper proteins, to • facilitate GDP/GTP exchange, or • promote GTP hydrolysis. A guanine nucleotide exchange factor (GEF) induces a conformational change that makes the nucleotidebinding 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). www. soran. edu. iq 4
A GTPase activating protein (GAP) causes a GTPbinding protein to hydrolyze its bound GTP to GDP + Pi. 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 www. soran. edu. iq 5
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. www. soran. edu. iq 6
Initiation of protein synthesis in E. coli requires initiation factors IF-1, IF-2, & IF-3. w IF-3 binds to the 30 S ribosomal subunit, freeing it from its complex with the 50 S subunit. w IF-1 assists binding of IF-3 to the 30 S ribosomal subunit. IF-1 also occludes the A site of the small ribosomal subunit, helping insure that the initiation aa-t. RNA f. Met -t. RNAf. Met can bind only in the P site & that no other aat. RNA can bind in the A site during initiation. w IF-2 is a small GTP-binding protein. IF-2 -GTP binds the initiator f. Met-t. RNAf. Met & helps it to dock with the small ribosome subunit. www. soran. edu. iq 7
w As m. RNA binds, IF-3 helps to correctly position the complex such that the t. RNAf. Met interacts via base pairing with the m. RNA initiation codon (AUG). A region of m. RNA upstream of the initiation codon, the Shine-Dalgarno sequence, base pairs with the 3' end of the 16 S r. RNA. This positions the 30 S ribosomal subunit in relation to the initiation codon. w 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. w Once the two ribosomal subunits come together, the m. RNA is threaded through a curved channel that wraps around the "neck" region of the small subunit. www. soran. edu. iq 8
Elongation cycle Ribosome structure and position of factors & t. RNAs based on cryo-EM with 3 D image reconstruction. Diagram provided by Dr. J. Frank, Wadsworth Center, NYS Dept. of Health. Partial images on subsequent slides are derived from this. Colors: large ribosome subunit, cyan; small subunit, pale yellow; EFTu, red; EF-G, blue. t. RNAs, gray, magenta, green, yellow, brown. www. soran. edu. iq 9
Elongation requires participation of elongation factors • EF-Tu (also called EF 1 A) • EF-Ts (EF 1 B) • EF-G (EF 2) EF-Tu & EF-G are small GTP-binding proteins. The sequence of events follows. www. soran. edu. iq 10
EF-Tu-GTP binds & delivers an aminoacyl-t. RNA to the A site on the ribosome. EF-Tu recognizes & binds all aminoacyl-t. RNAs with approx. the same affinity, when each t. RNA is bonded to the correct (cognate) amino acid. t. RNAs for different amino acids have evolved to differ slightly EF-Tu colored in structure, to compensate for different binding affinities of amino acid side-chains, so the aminoacyl-t. RNAs all have similar affinity for EF-Tu. www. soran. edu. iq 11
The t. RNA must have the correct anticodon to interact with the m. RNA codon positioned at the A site to form a base pair of appropriate geometry. Universally conserved bases of 16 S r. RNA 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 t. RNA has bound. Proofreading in part involves release of the aminoacylt. RNA prior to peptide bond formation, if the appropriate ribosomal conformation is not generated by this interaction. www. soran. edu. iq 12
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. www. soran. edu. iq 13
When EF-Tu delivers an aminoacyl-t. RNA to the ribosome, the t. RNA 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 t. RNA conformation relaxes, & the acceptor stem is repositioned to promote peptide bond formation. This process is called accommodation. www. soran. edu. iq EF-Tu colored 14
It includes rotation of the single-stranded 3' end of the acceptor stem of the A-site t. RNA 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 t. RNA, & adjacent to the mouth of the tunnel through which nascent polypeptides exit the ribosome. www. soran. edu. iq 15
EF-Ts functions as GEF to reactivate EF-Tu. 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. www. soran. edu. iq 16
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. www. soran. edu. iq 17
Compare, using Chime, the structures of: w EF-Tu with bound GDP w EF-Tu with bound GTP analog GDPNP w EF-Tu with bound GTP analog & Phe-t. RNAPhe 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-t. RNA, affect EF-Tu conformation more? www. soran. edu. iq 18
Transpeptidation (peptide bond formation) involves nucleophilic attack of the amino N of the amino acid linked to the 3'OH of the terminal adenosine of the t. RNA in the A site on the carbonyl C of the amino acid (with attached nascent polypeptide) in ester linkage to the t. RNA in the P site. The reaction is promoted by the geometry of the active site consisting solely of residues of the 23 S r. RNA of the large ribosomal subunit. No protein is found at the active site. www. soran. edu. iq 19
The 23 S r. RNA may be considered a "ribozyme. " As part of the reaction a proton (H+) is extracted from the attacking amino N. www. soran. edu. iq 20
This H+ is then donated to the hydroxyl of the t. RNA in the Pwww. soran. edu. iq site, as the ester linkage is cleaved. 21
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 peptidylt. RNA, along with ribose hydroxyls of active site r. RNA residues & structured water molecules that collectively form a H-bonded network at the active site. www. soran. edu. iq 22
The nascent polypeptide, one residue longer, is now linked to the A-site t. RNA. www. soran. edu. iq 23
However, translocation has already partly occurred, because peptide bond formation is associated with rotation of the single-stranded 3' end of the A-site t. RNA 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. www. soran. edu. iq 24
t. RNA grey, EF-Tu red, EF -G blue The unloaded t. RNA in the P site will shift to the E (exit) site during translocation. Translocation of the ribosome relative to m. RNA 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-t. RNA. 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. www. soran. edu. iq 25
The t. RNA with attached nascent polypeptide is pushed from the A site to the P site. Unloaded t. RNA that was in the P site shifts to the E site. Since t. RNAs are linked to m. RNA by codon-anticodon base pairing, the m. RNA moves relative to the ribosome. www. soran. edu. iq 26
Additionally, it has been postulated that translocation is spontaneous after peptide bond formation because: • the deacylated t. RNA in the P site has a higher affinity for the E site, & • the peptidyl-t. RNA 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. www. soran. edu. iq 27
The continued codon-anticodon base paring of the t. RNA 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 t. RNAs were to able to shift laterally by one base pair. Normally the empty t. RNA is released from the E site only after binding of the correct aminoacyl-t. RNA at the A site causes a decreased affinity for t. RNA in the E site Explore with Chime the 30 S moiety of a bacterial ribosome, complexed with a short, genetically engineered m. RNA, and with t. RNAPhe in the A, P, & E sites. www. soran. edu. iq 28
Chain termination requires release factors RF-1, 2, & RF-3 is a small GTP-binding protein. RF- w RF-1 & RF-2 recognize & bind to STOP codons. One or the other binds when a stop codon is reached. w RF-3 -GTP facilitates binding of RF-1 or RF-2 to the ribosome. www. soran. edu. iq 29
w Once release factors occupy the A site, Peptidyl Transferase catalyzes transfer of the peptidyl group to water (hydrolysis). w Hydrolysis of GTP on RF-3 causes a conformational change that results in dissociation of release factors. w A ribosomal recycling factor (RRF) is required, with EFG-GTP and IF-3, for release of uncharged t. RNA from the P site, and dissociation of the ribosome from m. RNA with separation of the two ribosomal subunits. www. soran. edu. iq 30
Eukaryotic Translation of m. RNA 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. w 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. w Mechanisms include regulation by signal-activated phosphorylation or dephosphorylation of initiation and elongation factors. www. soran. edu. iq 31
Translation of particular m. RNAs may be inhibited by small single-stranded micro. RNA molecules about 20 -22 nucleotides long. Micro. RNAs bind via base-pairing to 3' un-translated regions of m. RNA along with a protein complex RISC (RNAinduced silencing complex), inhibiting translation and in some cases promoting m. RNA degradation. w Tissue-specific expression of particular genomeencoded micro. RNAs is an essential regulatory mechanism controlling embryonic development. w Some forms of cancer are associated with altered expression of micro. RNAs that regulate synthesis of proteins relevant to cell cycle progression or apoptosis. www. soran. edu. iq 32
Protein factors that mediate & control translation are more numerous in eukaryotes than in prokaryotes. Eukaryotic factors are designated with the prefix "e". w Some factors are highly conserved across kingdoms. E. g. , the eukaryotic elongation factor e. EF 1 A is structurally and functionally similar to the prokaryotic EF-TU (EF 1 A). w In contrast, e. EF 1 B, the eukaryotic equivalent of the GEF EF-Ts, is relatively complex, having multiple subunits subject to regulatory phosphorylation. www. soran. edu. iq 33
Initiation of protein synthesis is much more complex in eukaryotes, & requires a large number of protein factors. Some eukaryotic initiation factors (e. g. , e. IF 3 & e. IF 4 G) serve as scaffolds, with multiple domains that bind other proteins during assembly of large initiation complexes. www. soran. edu. iq 34
Usually a pre-initiation complex forms, including: w several initiation factors w the small ribosomal subunit w the loaded initiator t. RNA, Met-t. RNAi. Met. This then binds to a separate complex that includes: w m. RNA w initiation factors including ones that interact with the 5' methylguanosine cap & the 3' poly-A tail, structures unique to eukaryotic m. RNA. w Within this complex m. RNA 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 Worm. Book. www. soran. edu. iq 35
After the initiation complex assembles, it translocates along the m. RNA in a process called scanning, until the initiation codon is reached. Scanning is facilitated by eukaryotic initiation factor e. IF 4 A, which functions as an ATP-dependent helicase to unwind m. RNA 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. www. soran. edu. iq 36
Some eukaryotic m. RNAs 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. www. soran. edu. iq 37
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