Last piece in the puzzle; the part that impacts directly on all cellular functions.
There are three general steps of translation:
1. initiation - events that happen before the first peptide bond is synthesized - including assembly of the ribosome on the mRNA.
2. elongation - events that happen between synthesis of the first and last peptide bonds.
3. termination - steps involving release of the completed polypeptide, disassembly of the ribosome from the mRNA.
Only between tRNA and mRNA on one hand and between tRNA and the amino acyl synthetase on the other, although there is some quality control during elongation. There are no other checks. The genetic code (as manifested in the codon and anticodon) determines which tRNA pairs with the mRNA. The amino acyl synthetase determines which amino acid is attached to which tRNA. At least one E. coli aa synthetase is RNA catalized. The first charged tRNA then interacts with the mRNA and small subunit of the ribosome, along with translation initiation factors.
Prokaryotic and eukaryotic different in details, but overall concept is similar.
(70 per large subunit, 40 per small subunit).
Synthesized mainly from genes in the nucleolus.
Transported to cytoplasm through nuclear pores.
(5S, 5.8S and 28S per large subunit; 18S per small subunit).
- S is abbreviation for Svedburg unit, which is a measure of the behavior of a molecule in density gradient centrifugation, related to size, shape and density, not molecular weight.
r RNAs are synthesized on amplified genes in the nucleolus.
The ribosomes are assembled and then transported to the cytoplasm (mainly as assembled subunits), through nuclear pores, although some remain in the nucleus.
The large subunit is 60S and the small subunit is 40S. The complete ribosome is 80S. Only the complete ribosome has the active sites for formation of a polypeptide from the genetic code.
Translation occurs either in the cytoplasm, on "free" ribosomes, or on the surface of the rough endoplasmic reticulum (RER).
Translation initiation plays an important role in control of gene expression. It allows fine tuning of protein synthesis and immediate responses to environmental changes. Moreover, it has been shown recently that deregulation of protein synthesis can lead to cell transformation in mammalian cells.
Initiation uses eukaryotic initiation factors (eIFs) 1, 2, 3, 4f and 5, mRNA, charged met tRNA, the small (40S) ribosomal subunit and GTP and ATP for energy. eIF2 binds to the charged met tRNA, the other factors assemble on the mRNA. This requires the use of energy (GTP, ATP) to make and break bonds and change conformations. eIF 1 and 3 interact with the 3' end of the 18S RNA in the small subunit. The Shine Delgarno sequence in the 5' untranslated leader of the mRNA disrupts the secondary structure at the 3' end of the 18S RNA and basepairs with it. eIF2 catalyses the addition of GTP to the initial charged tRNA complex. eIF3 stabilizes the interaction of the small subunit with the charged tRNA. The small subunit also recognizes the cap structure on the 5' end of the mRNA. The S1 protein helps to keep the mRNA single stranded. The 40S subunit then has its A site region over the start codon, and the charged tRNA anticodon pairs with the start codon. Next, the initiation complex is formed by the addition of the large (60S) subunit. The 3' end of the 18S RNA in the small subunit is complementary to the 28S RNA in the large subunit. The tRNA moves to the P site, the A site is open, a GTP is used up and initiation factors are released. At some point, a few bases are cut off the end of the poly A chain on the 3' end of the mRNA by PAN.There are at least 10 - 12 factors, others (not listed) are probably required also. Most have been characterized in yeast or rabbit reticulocyte systems.
| eIF2- | - binding tRNAiMet (3 proteins) |
| eIF4F | - multimer mRNA binding & unwinding; contains eIF4E, eIF4A, eIF4G (assembly protein with sites for eIF4A & eIF4E) can be influenced by outside factors/conditions |
| eIF4E | - 5' CAP binding (cdc33) can be influenced by outside factors/conditions |
| eIF4A | - mRNA binding, ATP binding, prototype DEAD Box protein, probable RNA helicase |
| eIF4B | - mRNA binding / unwinding |
| eIF3 | - mRNA binding, binds 40S subunit |
| eIF5 | - releases eIF2, eIF3 |
| eIF4C | - binds 60S subunit |
| eIF6 | - 40S to 60S joining can be influenced by outside factors/conditions |
| eIF1 | - mRNA binding, accessory |
| eIF4D | - unknown |
| eIF5A | - promotes first peptide bond? |
| eIF4G | - eIF4F assembly; also binds eIF3 and polyA binding protein (PABP) - which might interact with ribosomal subunits |
Two things happen in parallel: preparation of the mRNA for 40S binding, and preparation of the 40S subunit for mRNA binding.
Transcribed mRNA, following post-transcriptional modification, is transported from the nucleus to the cytoplasm or the rough endoplasmic reticulum (RER) where it engages with ribosomes and initiation factors to direct synthesis of specific polypeptides.
1. eIF4E (cap binding protein, CBP) binds the 5' cap (in mammals as part of the eIF4F complex).The start of translation is almost always at the AUG codon which specifies methionine (Met). The "scanning" model of translation sees the 40S subunit of the ribosome bind with the 5' cap with the help of initiation factors both in cis and trans (mRNAs without 5' caps are not translated efficiently in vitro), and then scans along the message until it encounters the first AUG site, where translation is initiated. The cap binding proteins are thought to unwind the leader sequence of the 5' UTS, thus removing secondary structure from ribosome binding sites. A few viral mRNAs (such as polio virus) are not capped, but can still be translated in vitro.
The 40S subunit will only stop scanning at the first AUG encountered if it is in the right context with the surrounding bases. If the 40S subunit does not bind, it is stabilized by joining with the 60S subunit. Protection assays show that the 40S subunit protects a region of up to 60 bases and the 60S subunit 30 - 40 bases.
An eukaryotic Translation Initiation Sequence (TIS) CCACCATGG has been identified in nuclear DNA. Mutations of the initiation codon exist, with a preponderance of Met -> Val substitutions through point mutation. A few rare cases of initiation codon have been found including GUG (Val), ACG (Thr), and CUG (Leu). Even if the initiation codon is AUG, the N-terminal amino acid of the mature polypeptide may not be methionine. Some examples of deletions which encompass or flank the initiation codon are known, e.g. deletion of -2 and -3 of the alpha 1 globin (HBA-1) gene; causing alpha thalassemia (a low level of α globin chains), by directly changing the consensus sequence at -3. This presumably prevents proper recognition of the of the initiation site. Mutation in translation initiation sites can also affect the levels of steady state mRNA present in a cell. The efficiency of translation can also be influenced by the secondary structure of the 5' leader sequence, particularly in extreme cases, as when hairpin loops are present.
With some mRNAs (such as those for ribosomal subunits, some elongation factors and PABP including some activated by insulin) with a 5' terminal oligopyrimidine (TOP) feature adjacent to the 5' cap, essential amino acid concentration in the cell causes (by an unknown pathway) phosphorylation of the TOP, by S6 kinase (which also phosphorylates ribosomal protein S6) which increases translation rates. Phosphorylation of eIF4E also increases translation rates (possibly by increasing its affinity for the 5' cap). This shows that there are many ways that cellular conditions can regulate translation initiation.
Translational initiation involves the positioning of the 40S small ribosomal subunit at the initiator codon and its association with the large 60S subunit, which is mediated by 12 eukaryotic initiation factors composed of approximately 25 polypeptides. For most cellular mRNAs, selection of the initiator AUG, followed by attaching the 60S subunit is preceded by a process best described as the "scanning" model. According to the model, the 40S subunit binds at the 5' end of the mRNA. This initial binding is mediated by eukaryotic translation initiation factors. The interaction of a 43S pre initiation complex {eIF3-40S-tRNA-eIF2-GTP} with eIF4G of the cap binding complex eIF4F is mediated by the multi - subunit factor eIF3, which is bound to the 40S subunit (see above). Once bound to the mRNA, the 43S pre initiation complex scans the 5' UTR until it locates the first AUG (see above). Recognition and successful positioning of the small ribosomal subunit at the initiator codon requires the presence of the ternary complex {eIF2-GTP-tRNAiMet} (see above).
The initiation factor eIF2 is composed of three subunits. This is the factor most often involved in general regulation of translation. Upon initiation, the eIF2A bound GTP is hydrolyzed to GDP. Recycling of eIF2A-GDP to eIF2A-GTP requires the activity of the guanine exchange factor eIF2B, itself composed of five subunits (a mutation in which leads to leukoencephalopathy, an inherited brain disease). This recycling is inhibited by phosphorylation of the eIF2α subunit (by gsk-3), thereby competitively inhibiting eIF2B and general translation initiation (this can be caused by starvation for essential amino acids - mechanism not known). The inhibition can be reversed in response to insulin dependent dephosphorylation. The ε subunit can also be phosphorylated. The phosphorylation of eIF2A is mediated by the protein kinases Gcn2p (yeast) and PKR (mammalian), as well as others.
The factor eIF4E, or cap binding protein (CBP), binds to the cap structure (m7GpppNp). This association of eIF4G with eIF4E in mammalian cells is competitively inhibited by a series of 4E - binding proteins (4E-BPs 1 - 3), which then regulate cap-dependent translation initiation by blocking the eIF4G binding site. This can be stoped by phosphorylation of 4E-BP1, due to essential amino acid concentration.
Studies have also shown that eukaryotic ribosomes can reinitiate translation by inhibiting disassembly through trans acting factors.
Although the large majority of mRNAs are capped at their 5' ends, some (growth factors VEGF, FGF2 and PDGF; apoptosis associated gene products Apaf-1, IXAP; transcription factors c-myc; the potassium channel Kv1.4 and others) are not. This includes some plus strand RNA virus mRNAs (polio virus, human rhinovirus, hepatitis A and hepatitis C viruses). Also, two thirds of cell proliferation mRNAs and proto oncogene mRNAs contain atypical 5' UTRs, which might include them in this category. In addition, many plant messengers are uncapped.
These mRNAs contain atypically long (200bp) 5' UTRs or have more than one AUG codon. They are translated by cap independent internal initiation. In this type of translation, the ribosome binds to an internal RNA sequence near the initiator AUG. The sequences that form this element are known as the Internal Ribosome Entry Site (IRES) and the process is called IRES mediated translation.
IRES structure probably contains conserved elements, and IRES point mutations generally down regulate translation, but there are probably different types of IRES elements with different structures and which bind different factors. Many IRES types require initiation factors to initiate translation. One group requires eIF4G and associated factors, another requires eIF2 associated factors but not eIF4G, another group doesn't require either eIF2 or GTP, while yet another group seems to interact directly with rRNA. Different regions of the IRES seem to have different functions, such as binding to eIFs or for RNA - RNA interactions, etc. Messages that contain both a cap and an IRES element can be translated normally, but this occurs at a low level (due to secondary structure?) and produces, in some cases, an atypical protein product. In these cases, possibly the IRES translation is the "normal" mode of translation. In general, binding to the IRES may also induce conformational changes in the small ribosomal subunit.
Some cellular IRES transcription is mediated by specific cell physiological states, such as apoptosis, hypoxia, vascular lesions, γ-irradiation, growth arrest and angiogenisis, but the factors which mediate this stimulation are not known.
Finding the trasnlation start site seems to be a two part system (as in the normal system) with structures aurrounding the AUG codon as one part and the IRES (taking the place of the 5' cap and some initiation factors?) as the other. As mentioned above, there seem to be a range of factors needed for these recognitions, depending on the type of IRES, etc.. In "normal" transcription, both the polyA tail and the 5' cap are involved in transcription, forming a cyclized product. In IRES mediated translation, this is not clear. Some cases seem to involve the polyA tail, but some do not. Other factors may be needed for cyclization, or it may not occur.
Yeasts contain a short RNA that inhibits IRES binding. Many of the mRNAs with long, structured 5' UTRs actually code for regulatory proteins. They seem to have a need for other factors, apart fromthe normal eIFs. The DExH box protein DHX29 (which has helicase activity) is required for their translation. It binds to the 40 S subunit, causing conformational changes in it, and hydrolizes NTPs (stimulated by the 43 S complex), which is necessary for the formation of the 48 S active complex.
IRES mediated translation may be a system that has evolved to translate specific proteins in specific (stress?) conditionse.
Ferritin is an iron binding protein. mRNA encoding ferritin heavy and light chains contains 5"UTR stem - loop structures which function as iron responsive elements (IREs) to which an iron regulatory protein binds when cells are low in iron. There are two known IRPs, IRP1 (98 k dal) and IRP2 (105 k dal), which are 57% homologous. These IRPs show tissue specific expression. Binding of the IRP to the IRE blocks eIF4G - eIF3 interaction and inhibits translation. IRPs are iron containing proteins, with a [4S - 4Fe] cluster, and acts as an enzyme, aconitase. When cellular iron levels are low, one of the four iron atoms in the cluster is lost. The [4s - 4Fe] cluster disassembles, aconitase activity is lost and RNA binding (at the IRE) is gained.
Elongation requires eukaryotic elongation factors (eEFs) 1, 1β, 2 and GTP for energy.
eEF1 bound to GDP complexes with eEF1β. This gives the complex a higher affinity for GTP, and it exchanges its GDP for a GTP. eEF1β then departs the complex. eEF1 binds to the TψCG loop of a charged tRNA. The whole complex is then taken into the A site, ant if it fits, the GTP is hydrolixed to GDP, the amino acid attached to the tRNA is added to the nacent polypeptide, and the GDP-eEF1 complex comes out of the A site.
The GTP hydorolysis above drives the translocation process, whereby the incoming charged tRNA is positioned, the growing polypeptide chain, the chain is transfered to the next amino acid on the new tRNA and the new tRNA now carrying the polypeptide chain moves to the P site. The process seems to have two distinct components; a pause (on average about 2 seconds, but up to several minutes is possible), followed by a movement step -in three rapid pieces - to the next codon, which takes about 0.1 second. This continues until a termination (nonsense) site is reached. There is a sort of proofreading in this prosess, where mismatches in codon - anitcodon pairing during chain elongation bring about chain termination through the use of the termination factors. The precise method isn't known, but possibility is that conformational changes induced by the mispairing lead to higher termination activity. The result of this is that there is less than 1 mistake/105 amino acids.
Since the stop codon doesn't code for an amino acid, the system pauses. The termination factor interacts withe the A site and changes the activity of the peptidyl transferase, which removes (hydrolyses) the polupeptide chain from the last tRNA, and the ribosome disassembles. This requires use of a GTP for energy. In prokaryotes, part of the termination complex, Rf1 lies on top of the stop codon, and the rest of it interacts with the 16 and 23S RNAs of the ribosome, inducing a conformational change which leads to cleavage of the polypeptide chain from the terminal tRNA.
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