EF-4

Last updated
Elongation factor 4
Identifiers
SymbolEF-4
InterPro IPR006297
For well-annotaterd examples, see P60785 (E. coli LepA) and Q8N442 (human GUF1).

Elongation factor 4 (EF-4) is an elongation factor that is thought to back-translocate on the ribosome during the translation of RNA to proteins. It is found near-universally in bacteria [1] and in eukaryotic endosymbiotic organelles including the mitochondria and the plastid. [2] [3] Responsible for proofreading during protein synthesis, EF-4 is a recent addition to the nomenclature of bacterial elongation factors. [4]

Contents

Prior to its recognition as an elongation factor, EF-4 was known as leader peptidase A (LepA), as it is the first cistron on the operon carrying the bacterial leader peptidase. In eukaryotes it is traditionally called GUF1 (GTPase of Unknown Function 1). [5] It has the preliminary EC number 3.6.5.n1. [6]

Evolutionary background

LepA has a highly conserved sequence. LepA orthologs have been found in bacteria and almost all eukaryotes. The conservation in LepA has been shown to cover the entire protein. More specifically, the amino acid identity of LepA among bacterial orthologs ranges from 55%-68%. [4]

Two forms of LepA have been observed; one form of LepA branches with mitochondrial LepA sequences, while the second form branches with cyanobacterial orthologs. These findings demonstrate that LepA is significant for bacteria, mitochondria, and plastids. LepA is absent from archaea. [4]

Structure

The gene encoding LepA is known to be the first cistron as part of a bicistron operon. LepA is a polypeptide of 599 amino acids with a molecular weight of 67 kDa. The amino acid sequence of LepA indicates that it is a G protein, which consists of five known domains. The first four domains are strongly related to domains I, II, III, and V of primary elongation factor EF-G. However, the last domain of LepA is unique. This specific domain resides on the C-terminal end of the protein structure. [7] This arrangement of LepA has been observed in the mitochondria of yeast cells to human cells.

Function

LepA is suspected to improve the fidelity of translation by recognizing a ribosome with mistranslocated tRNA and consequently inducing a back-translocation. By back-translocating the already post-transcriptionally modified ribosome, the EF-G factor capable of secondary translocation. Back-translocation by LepA occurs at a similar rate as an EF-G-dependent translocation. As mentioned above, EF-G's structure is highly analogous to LepA's structure; LepA's function is thus similarly analogous to EF-G's function. However, Domain IV of EF-G has been shown through several studies to occupy the decoding sequence of the A site after the tRNAs have been translocated from A and P sites to the P and E sites. Thus, domain IV of EF-G prevents back-movement of the tRNA. Despite the structural similarities between LepA and EF-G, LepA lacks this Domain IV. Thus LepA reduces the activation barrier between Pre and POST states in a similar way to EF-G but is, at the same time, able to catalyze a back-translocation rather that a canonical translocation.

Activity

LepA exhibits uncoupled GTPase activity. This activity is stimulated by the ribosome to the same extent as the activity of EF-G, which is known to have the strongest ribosome-dependent GTPase activity among all characterized G proteins involved in translation. Conversely, uncoupled GTPase activity occurs when the ribosome stimulation of GTP cleavage is not directly dependent on protein synthesis. In the presence of GTP, LepA works catalytically. On the other hand, in the presence of the nonhydrolysable GTP – GDPNP – the LepA action becomes stoichiometric, saturating at about one molecule per 70S ribosomes. This data demonstrates that GTP cleavage is required for dissociation of LepA from the ribosome, which is demonstrative of a typical G protein. At low concentrations of LepA (less than or equal to 3 molecules per 70S ribosome), LepA specifically recognizes incorrectly translocated ribosomes, back-translocates them, and thus allows EF-G to have a second chance to catalyze the correct translocation reaction. At high concentrations (about 1 molecule per 70S ribosome), LepA loses its specificity and back-translocates every POST ribosome. This places the translational machinery in a nonreproductive mode. This explains the toxicity of LepA when it is found in a cell in high concentrations. Hence, at low concentrations LepA significantly improves the yield and activity of synthesized proteins; however, at high concentrations LepA is toxic to cells.

Additionally, LepA has an effect on peptide bond formation. Through various studies in which functional derivatives of ribosomes were mixed with puromycin (an analog of the 3' end of an aa-tRNA) it was determined that adding LepA to a post transcriptionally modified ribosome prevents dipeptide formation as it inhibits the binding of aa-tRNA to the A site.

Experimental data

There have been various experiments elucidating the structure and function of LepA. One notable study is termed the "toeprinting experiment": this experiment helped to determine LepA's ability to back-translocate. In this case, a primer was extended via reverse transcription along mRNA which was ribosome-bound. The primers from modified mRNA strands from various ribosomes were extended with and without LepA. An assay was then conducted with both PRE and POST states, and cleavage studies revealed enhanced positional cleavage in the POST state as opposed to the PRE state. Since the POST state had been in the presence of LepA (plus GTP), it was determined that the strong signal characteristic of the POST state was the result of LepA which then brought the signal down to the level of the PRE state. Such a study demonstrated that that ribosome, upon binding to the LepA-GTP complex assumes the PRE state configuration.

See also

Related Research Articles

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The signal recognition particle (SRP) is an abundant, cytosolic, universally conserved ribonucleoprotein that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recapping.

Initiation factors are proteins that bind to the small subunit of the ribosome during the initiation of translation, a part of protein biosynthesis.

A release factor is a protein that allows for the termination of translation by recognizing the termination codon or stop codon in an mRNA sequence. They are named so because they release new peptides from the ribosome.

<span class="mw-page-title-main">Elongation factor</span> Proteins functioning in translation

Elongation factors are a set of proteins that function at the ribosome, during protein synthesis, to facilitate translational elongation from the formation of the first to the last peptide bond of a growing polypeptide. Most common elongation factors in prokaryotes are EF-Tu, EF-Ts, EF-G. Bacteria and eukaryotes use elongation factors that are largely homologous to each other, but with distinct structures and different research nomenclatures.

<span class="mw-page-title-main">EF-Tu</span> Prokaryotic elongation factor

EF-Tu is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes. It is found in eukaryotic mitochondria as TUFM.

Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the initiation phase of eukaryotic translation. These proteins help stabilize the formation of ribosomal preinitiation complexes around the start codon and are an important input for post-transcription gene regulation. Several initiation factors form a complex with the small 40S ribosomal subunit and Met-tRNAiMet called the 43S preinitiation complex. Additional factors of the eIF4F complex recruit the 43S PIC to the five-prime cap structure of the mRNA, from which the 43S particle scans 5'-->3' along the mRNA to reach an AUG start codon. Recognition of the start codon by the Met-tRNAiMet promotes gated phosphate and eIF1 release to form the 48S preinitiation complex, followed by large 60S ribosomal subunit recruitment to form the 80S ribosome. There exist many more eukaryotic initiation factors than prokaryotic initiation factors, reflecting the greater biological complexity of eukaryotic translation. There are at least twelve eukaryotic initiation factors, composed of many more polypeptides, and these are described below.

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<span class="mw-page-title-main">EF-G</span> Prokaryotic elongation factor

EF-G is a prokaryotic elongation factor involved in protein translation. As a GTPase, EF-G catalyzes the movement (translocation) of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome.

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EF-Ts is one of the prokaryotic elongation factors. It is found in human mitochondria as TSFM. It is similar to eukaryotic EF-1B.

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<span class="mw-page-title-main">Elongation factor P</span>

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References

  1. ↑ Margus, Tõnu; Remm, Maido; Tenson, Tanel (December 2007). "Phylogenetic distribution of translational GTPases in bacteria". BMC Genomics. 8 (1): 15. doi: 10.1186/1471-2164-8-15 . PMC   1780047 . PMID   17214893.
  2. ↑ Youngman EM, Green R (February 2007). "Ribosomal translocation: LepA does it backwards". Curr. Biol. 17 (4): R136–9. doi: 10.1016/j.cub.2006.12.029 . PMID   17307049.
  3. ↑ March PE, Inouye M (November 1985). "GTP-binding membrane protein of Escherichia coli with sequence homology to initiation factor 2 and elongation factors Tu and G". Proc. Natl. Acad. Sci. U.S.A. 82 (22): 7500–4. Bibcode:1985PNAS...82.7500M. doi: 10.1073/pnas.82.22.7500 . PMC   390844 . PMID   2999765.
  4. 1 2 3 Qin Y, Polacek N, Vesper O, et al. (November 2006). "The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome". Cell. 127 (4): 721–33. doi: 10.1016/j.cell.2006.09.037 . PMID   17110332.
  5. ↑ Bauerschmitt, Heike; Funes, Soledad; Herrman, Johannes (June 2008). "The Membrane-bound GTPase Guf1 Promotes Mitochondrial Protein Synthesis under Suboptimal Conditions*". Journal of Biological Chemistry. 234 (25): 17139–17146. doi: 10.1074/jbc.M710037200 . PMID   18442968.
  6. ↑ "ENZYME entry: EC 3.6.5.n1" . Retrieved October 21, 2021.
  7. ↑ Evans RN, Blaha G, Bailey S, Steitz TA (March 2008). "The structure of LepA, the ribosomal back translocase". Proc. Natl. Acad. Sci. U.S.A. 105 (12): 4673–8. Bibcode:2008PNAS..105.4673E. doi: 10.1073/pnas.0801308105 . PMC   2290774 . PMID   18362332.
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