Directed evolution

From Wikipedia, the free encyclopedia

An example of directed evolution with comparison to natural

evolution. The inner cycle indicates the 3 stages of the directed

evolution cycle with the natural process being mimicked in

brackets. The outer circle demonstrates steps a typical

experiment. The red symbols indicate functional variants, the

pale symbols indicate variants with reduced function.

Directed evolution (DE, "gelenkte Evolution") is a method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids toward a user-defined goal.^[1] It consists of subjecting a gene to iterative rounds of mutagenesis (creating a library of variants), selection (expressing the variants and isolating members with the desired function), and amplification (generating a template for the next round). It can be performed in vivo (in living cells), or in vitro (free in solution or microdroplet). Directed evolution is used both for protein engineering as an alternative to rationally designing modified proteins, as well as studies of fundamental evolutionary principles in a controlled, laboratory environment.

Principles

Directed evolution is analogous to climbing a hill on a 'fitness landscape' where elevation represents the desired property. Each round of selection samples mutants on all sides of the starting template (1) and selects the mutant with the highest elevation, thereby climbing the hill. This is repeated until a local summit is reached (2).

Directed evolution is a mimic of the natural evolution cycle in a laboratory setting. Evolution requires three things to happen: variation between replicators, that the variation causes fitness differences upon which selection acts, and that this variation is heritable. In DE, a single gene is evolved by iterative rounds of mutagenesis, selection or screening, and amplification.^[2] Rounds of these steps are typically repeated, using the best variant from one round as the template for the next to achieve stepwise improvements.

The likelihood of success in a directed evolution experiment is directly related to the total library size, as evaluating more mutants increases the chances of finding one with the desired properties.^[3]

Generating variation

Starting gene (left) and library of variants (right). Point mutations change single nucleotides. Insertions and deletions add or remove sections of DNA. Shuffling recombines segments of two (or more) similar genes.

The first step in performing a cycle of directed evolution is the generation of a library of variant genes. The sequence space for random sequence is vast (10¹³⁰ possible sequences for a 100 amino acid protein) and extremely sparsely populated by functional proteins. Neither experimental,^[4] nor natural^{[5][not in citation given]} evolution can ever get close to sampling so many sequences. Of course, natural evolution samples variant sequences close to functional protein sequences and this is imitated in DE by mutagenising an already functional gene. Some calculations suggest it is entirely feasible that for all practical (i.e. functional and structural) purposes, protein sequence space has been fully explored during the course of evolution of life on Earth.^[6]

The starting gene can be mutagenised by random point mutations (by chemical mutagens or error prone PCR)^[7][8] and insertions and deletions (by transposons).^[9] Gene recombination can be mimicked by DNA shuffling^[10][11] of several sequences (usually of more than 70% homology) to jump into regions of sequence space between the shuffled parent genes. Finally, specific regions of a gene can be systematically randomised^[12] for a more focused approach based on structure and function knowledge. Depending on the method, the library generated will vary in the proportion of functional variants it contains. Even if an organism is used to express the gene of interest, by mutagenising only that gene, the rest of the organism’s genome remains the same and can be ignored for the evolution experiment (to the extent of providing a constant genetic environment).

Detecting fitness differences

The majority of mutations are deleterious and so libraries of mutants tend to mostly have variants with reduced activity.^[13] Therefore, a high-throughput assay is vital for measuring activity to find the rare variants with beneficial mutations that improve the desired properties. Two main categories of method exist for isolating functional variants. Selection systems directly couple protein function to survival of the gene, whereas screening systems individually assay each variant and allow a quantitative threshold to be set for sorting a variant or population of variants of a desired activity. Both selection and screening can be performed in living cells (in vivo evolution) or performed directly on the protein or RNA without any cells (in vitro evolution).^[14][15]

During in vivo evolution, each cell (usually bacteria or yeast) is transformed with a plasmid containing a different member of the variant library. In this way, only the gene of interest differs between the cells, with all other genes being kept the same. The cells express the protein either in their cytoplasm or surface where its function can be tested. This format has the advantage of selecting for properties in a cellular environment, which is useful when the evolved protein or RNA is to be used in living organisms. When performed without cells, DE involves using in vitro transcription translation to produce proteins or RNA free in solution or compartmentalised in artificial microdroplets. This method has the benefits of being more versatile in the selection conditions (e.g. temperature, solvent), and can express proteins that would be toxic to cells. Furthermore, in vitro evolution experiments can generate far larger libraries (up to 10¹⁵) because the library DNA need not be inserted into cells (often a limiting step).

Selection

Selection for binding activity is conceptually simple. The target molecule is immobilised on a solid support, a library of variant proteins is flowed over it, poor binders are washed away, and the remaining bound variants recovered to isolate their genes.^[16] Binding of an enzyme to immobilised covalent inhibitor has been also used as an attempt to isolate active catalysts. This approach, however, only selects for single catalytic turnover and is not a good model of substrate binding or true substrate reactivity. If an enzyme activity can be made necessary for cell survival, either by synthesizing a vital metabolite, or destroying a toxin, then cell survival is a function of enzyme activity.^[17][18] Such systems are generally only limited in throughput by the transformation efficiency of cells. They are also less expensive and labour-intensive than screening, however they are typically difficult to engineer, prone to artefacts and give no information on the range of activities present in the library.

Screening

An alternative to selection is a screening system. Each variant gene is individually expressed and assayed to quantitatively measure the activity (most often by a colourgenic or fluorogenic product). The variants are then ranked and the experimenter decides which variants to use as templates for the next round of DE. Even the most high throughput assays usually have lower coverage than selection methods but give the advantage of producing detailed information on each one of the screened variants. This disaggregated data can also be used to characterise the distribution of activities in libraries which is not possible in simple selection systems. Screening systems, therefore, have advantages when it comes to experimentally characterising adaptive evolution and fitness landscapes.

Ensuring heredity

An expressed protein can either be covalently linked to its gene (as in mRNA, left) or compartmentalized with it (cells or artificial compartments, right). Either way ensures that the gene can be isolated based on the activity of the encoded protein.

When functional proteins have been isolated, it is necessary that their genes are too, therefore a genotype-phenotype link is required.^[19] This can be covalent, such as mRNA display where the mRNA gene is linked to the protein at the end of translation by puromycin.^[20] Alternatively the protein and its gene can be co-localised by compartmentalisation in living cells^[21] or emulsion droplets.^[22] The gene sequences isolated are then amplified by PCR or by transformed host bacteria. Either the single best sequence, or a pool of sequences can be used as the template for the next round of mutagenesis. The repeated cycles of Diversification-Selection-Amplification generate protein variants adapted to the applied selection pressures.

Comparison to rational protein design

Advantages of directed evolution

Rational design of a protein relies on an in-depth knowledge of the protein structure, as well as its catalytic mechanism.^[23][24] Specific changes are then made by site-directed mutagenesis in an attempt to change the function of the protein. A drawback of this is that even when the structure and mechanism of action of the protein are well known, the change due to mutation is still difficult to predict. Therefore, an advantage of DE is that there is no need to understand the mechanism of the desired activity or how mutations would affect it.^[25]

Limitations of directed evolution

A restriction of directed evolution is that a high-throughput assay is required in order to measure the effects of a large number of different random mutations. This can require extensive research and development before it can be used for directed evolution. Additionally, such assays are often highly specific to monitoring a particular activity and so are not transferable to new DE experiments.^[26]

Additionally, selecting for improvement in the assayed function simply generates improvements in the assayed function. To understand how these improvements are achieved, the properties of the evolving enzyme have to be measured. Improvement of the assayed activity can be due to improvements in enzyme catalytic activity or enzyme concentration. There is also no guarantee that improvement on one substrate will improve activity on another. This is particularly important when the desired activity cannot be directly screened or selected for and so a ‘proxy’ substrate is used. DE can lead to evolutionary specialisation to the proxy without improving the desired activity. Consequently, choosing appropriate screening or selection conditions is vital for successful DE.

Combinatorial approaches

Combined, 'semi-rational' approaches are being investigated to address the limitations of both rational design and directed evolution.^[27][28] Beneficial mutations are rare, so large numbers of random mutants have to be screened to find improved variants. 'Focussed libraries' concentrate on randomising regions thought to be richer in beneficial mutations for the mutagenesis step of DE. A focussed library contains fewer variants than a traditional random mutagenesis library and so does not require such high-throughput screening.

Creating a focussed library requires some knowledge of which residues in the structure to mutate. For example, knowledge of the active site of an enzyme may allow just the residues known to interact with the substrate to be randomised.^[29][30] Alternatively, knowledge of which protein regions are variable in nature can guide mutagenesis in just those regions.^[31][32]

Uses

Directed evolution is frequently used for protein engineering as an alternative to rational design,^[33] but can also be used to investigate fundamental questions of enzyme evolution.^[34]

Protein engineering

As a protein engineering tool, DE has been most successful in three areas:

1. Improving protein stability for biotechnological use at high temperatures or in harsh solvents.^[35][36]
2. Improving binding affinity of therapeutic antibodies (Affinity maturation)^[37] and the activity of de novo designed enzymes.^[38]
3. Altering substrate specificity of existing enzymes,^{[39][40][41][42]} (often for use in industry).^[43]

Evolution studies

The study of natural evolution is traditionally based on extant organisms and their genes. However, research is fundamentally limited by the lack of fossils (and particularly the lack of ancient DNA sequences)^[44][45] and incomplete knowledge of ancient environmental conditions. Directed evolution investigates evolution in a controlled system of genes for individual enzymes,^[46][47][48] ribozymes^[49] and replicators^[50][51] (similar to experimental evolution of eukaryotes,^[52][53] prokaryotes^[54] and viruses^[55]).

DE allows control of selection pressure, mutation rate and environment (both the abiotic environment such as temperature, and the biotic environment, such as other genes in the organism). Additionally, there is a complete record of all evolutionary intermediate genes. This allows for detailed measurements of evolutionary processes, for example epistasis, evolvability, adaptive constraint^[56][57] fitness landscapes,^[58] and neutral networks.^[59]

Translation (biology)

From Wikipedia, the free encyclopedia

 

Overview of the translation of eukaryotic messenger RNA

Diagram showing the translation of mRNA and the synthesis of proteins by a ribosome

In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is called gene expression.

In translation, messenger RNA (mRNA) is decoded in a ribosome to produce a specific amino acid chain, or polypeptide. The polypeptide later folds into an active protein and performs its functions in the cell. The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome.

Translation proceeds in three phases:

1. Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.
2. Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon. The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain.
3.  
4. The three phases of translation initiation polymerase binds to the DNA strand and moves along until the small ribosomal subunit binds to the DNA. Elongation is initiated when the large subunit attaches and termination end the process of elongation. Termination: When a stop codon is reached, the ribosome releases the polypeptide.

In prokaryotes (bacteria), translation occurs in the cytoplasm, where the large and small subunits of the ribosome bind to the mRNA. In eukaryotes, translation occurs in the cytosol or across the membrane of the endoplasmic reticulum in a process called co-translational translocation. In co-translational translocation, the entire ribosome/mRNA complex binds to the outer membrane of the rough endoplasmic reticulum (ER) and the new protein is synthesized and released into the ER; the newly created polypeptide can be stored inside the ER for future vesicle transport and secretion outside the cell, or immediately secreted.

Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA, do not undergo translation into proteins.

A number of antibiotics act by inhibiting translation. These include anisomycin, cycloheximide,  chloramphenicol, tetracycline, streptomycin, erythromycin, and puromycin. Prokaryotic ribosomes have a different structure from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial infections without any harm to a eukaryotic host's cells.

Basic mechanisms

A ribosome translating a protein that is secreted into the endoplasmic reticulum. tRNAs are colored dark blue.

Tertiary structure of tRNA. CCA tail in yellow, Acceptor stem in purple, Variable loop in orange, D arm in red, Anticodon arm in blue with Anticodon in black, T arm in green.

The basic process of translation is the addition of one amino acid at a time to the end of the polypeptide being formed. This process takes place inside the ribosome. A ribosome is made up of two subunits, a small 40S subunit and a large 60S subunit. These subunits come together before translation of mRNA into a protein to provide a location for translation to be carried out and a polypeptide to be produced.^[1] The choice of amino acid type to be added is determined by the genetic code on the mRNA molecule. Each amino acid added is matched to a three nucleotide subsequence of the mRNA. For each such triplet possible, the corresponding amino acid is accepted. The successive amino acids added to the chain are matched to successive nucleotide triplets in the mRNA. In this way, the sequence of nucleotides in the template mRNA chain determines the sequence of amino acids in the generated polypeptide.^[2] Addition of an amino acid occurs at the C-terminus of the peptide and thus translation is said to be amino-to-carboxyl directed.^[3]

The mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to the ribosomes. The ribonucleotides are "read" by translational machinery in a sequence of nucleotide triplets called codons. Each of those triplets codes for a specific amino acid.

The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a multisubunit structure containing rRNA and proteins. It is the "factory" where amino acids are assembled into proteins. tRNAs are small noncoding RNA chains (74-93 nucleotides) that transport amino acids to the ribosome. tRNAs have a site for amino acid attachment, and a site called an anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes for their cargo amino acid.

Aminoacyl tRNA synthetases (enzymes) catalyze the bonding between specific tRNAs and the amino acids that their anticodon sequences call for. The product of this reaction is an aminoacyl-tRNA. In prokaryotes, this aminoacyl-tRNA is carried to the ribosome by EF-Tu, where mRNA codons are matched through complementary base pairing to specific tRNA anticodons. Aminoacyl-tRNA synthetases that mispair tRNAs with the wrong amino acids can produce mischarged aminoacyl-tRNAs, which can result in inappropriate amino acids at the respective position in protein. This "mistranslation"^[4] of the genetic code naturally occurs at low levels in most organisms, but certain cellular environments cause an increase in permissive mRNA decoding, sometimes to the benefit of the cell.

The ribosome has three sites for tRNA to bind. They are the aminoacyl site (abbreviated A), the peptidyl site (abbreviated P) and the exit site (abbreviated E). With respect to the mRNA, the three sites are oriented 5’ to 3’ E-P-A, because ribosomes move toward the 3' end of mRNA. The A-site binds the incoming tRNA with the complementary codon on the mRNA. The P-site holds the tRNA with the growing polypeptide chain. The E-site holds the tRNA without its amino acid. When an aminoacyl-tRNA initially binds to its corresponding codon on the mRNA, it is in the A site. Then, a peptide bond forms between the amino acid of the tRNA in the A site and the amino acid of the charged tRNA in the P site. The growing polypeptide chain is transferred to the tRNA in the A site. Translocation occurs, moving the tRNA in the P site, now without an amino acid, to the E site; the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P site. The tRNA in the E site leaves and another aminoacyl-tRNA enters the A site to repeat the process."^[5]

After the new amino acid is added to the chain, and after the mRNA is released out of the nucleus and into the ribosome's core, the energy provided by the hydrolysis of a GTP bound to the translocase EF-G (in prokaryotes) and eEF-2 (in eukaryotes) moves the ribosome down one codon towards the 3' end. The energy required for translation of proteins is significant. For a protein containing n amino acids, the number of high-energy phosphate bonds required to translate it is 4n-1^{[citation needed]}. The rate of translation varies; it is significantly higher in prokaryotic cells (up to 17-21 amino acid residues per second) than in eukaryotic cells (up to 6-9 amino acid residues per second).^[6]

Even though the ribosomes are usually considered accurate and processive machines, the translation process is subject to errors that can lead either to the synthesis of erroneous proteins or to the premature abandonment of translation. The rate of error in synthesizing proteins has been estimated to be between 1/10⁵ and 1/10³ misincorporated amino acids, depending on the experimental conditions.^[7] The rate of premature translation abandonment, instead, has been estimated to be of the order of magnitude of 10⁻⁴ events per translated codon.^[8] The correct amino acid is covalently bonded to the correct transfer RNA (tRNA) by amino acyl transferases. The amino acid is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, the tRNA is termed "charged". Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with the help of initiation factors (IF). In prokaryotes, initiation of protein synthesis involves the recognition of a purine-rich initiation sequence on the mRNA called the Shine-Delgarno sequence. The Shine-Delgarno sequence binds to a complementary pyrimidine-rich sequence on the 3' end of the 16S rRNA part of the 30S ribosomal subunit. The binding of these complementary sequences ensures that the 30S ribosomal subunit is bound to the mRNA and is aligned such that the initiation codon is placed in the 30S portion of the P-site. Once the mRNA and 30S subunit are properly bound, an initiation factor brings the initiator tRNA-amino acid complex, f-Met-tRNA, to the 30S P site. The initiation phase is completed once a 50S subunit joins the 30 subunit, forming an active 70S ribosome.^[9] Termination of the polypeptide happens when the A site of the ribosome faces a stop codon (UAA, UAG, or UGA) on the mRNA. tRNA usually cannot recognize or bind to stop codons. Instead, the stop codon induces the binding of a release factor protein that prompts the disassembly of the entire ribosome/mRNA complex and the hydrolysis and the release of the polypeptide chain from the ribosome. Drugs or special sequence motifs on the mRNA can change the ribosomal structure so that near-cognate tRNAs are bound to the stop codon instead of the release factors. In such cases of 'translational readthrough', translation continues until the ribosome encounters the next stop codon.^[10]

The process of translation is highly regulated in both eukaryotic and prokaryotic organisms. Regulation of translation can impact the global rate of protein synthesis which is closely coupled to the metabolic and proliferative state of a cell. In addition, recent work has revealed that genetic differences and their subsequent expression as mRNAs can also impact translation rate in an RNA-specific manner.^[11]

Genetic code

Whereas other aspects such as the 3D structure, called tertiary structure, of protein can only be predicted using sophisticated algorithms, the amino acid sequence, called primary structure, can be determined solely from the nucleic acid sequence with the aid of a translation table.

This approach may not give the correct amino acid composition of the protein, in particular if unconventional amino acids such as selenocysteine are incorporated into the protein, which is coded for by a conventional stop codon in combination with a downstream hairpin (SElenoCysteine Insertion Sequence, or SECIS).

There are many computer programs capable of translating a DNA/RNA sequence into a protein sequence. Normally this is performed using the Standard Genetic Code, however, few programs can handle all the "special" cases, such as the use of the alternative initiation codons. For instance, the rare alternative start codon CTG codes for Methionine when used as a start codon, and for Leucine in all other positions.

Example: Condensed translation table for the Standard Genetic Code (from the NCBI Taxonomy webpage).

 AAs    = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG
 Starts = ---M---------------M---------------M----------------------------
 Base1  = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
 Base2  = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
 Base3  = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG

Translation tables

Even when working with ordinary eukaryotic sequences such as the Yeast genome, it is often desired to be able to use alternative translation tables—namely for translation of the mitochondrial genes. Currently the following translation tables are defined by the NCBI Taxonomy Group for the translation of the sequences in GenBank:

1. The Standard
2. The Vertebrate Mitochondrial Code
3. The Yeast Mitochondrial Code
4. The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code
5. The Invertebrate Mitochondrial Code
6. The Ciliate, Dasycladacean and Hexamita Nuclear Code
9. The Echinoderm and Flatworm Mitochondrial Code
10. The Euplotid Nuclear Code
11. The Bacterial and Plant Plastid Code
12. The Alternative Yeast Nuclear Code
13. The Ascidian Mitochondrial Code
14. The Alternative Flatworm Mitochondrial Code
15. Blepharisma Nuclear Code
16. Chlorophycean Mitochondrial Code
21. Trematode Mitochondrial Code
22. Scenedesmus obliquus mitochondrial Code
23. Thraustochytrium Mitochondrial Code

Ribosome

From Wikipedia, the free encyclopedia

Cell biology
The animal cell
Components of a typical animal cell: Nucleolus Nucleus Ribosome (little dots) Vesicle Rough endoplasmic reticulum Golgi apparatus (or "Golgi body") Cytoskeleton Smooth endoplasmic reticulum Mitochondrion Vacuole Cytosol (fluid that contains organelles, comprising the cytoplasm) Lysosome Centrosome Cell membrane

Figure 1: Ribosomes assemble polymeric protein molecules whose sequence is controlled by the sequence of messenger RNA molecules. This is required by all living cells and associated viruses.

The ribosome (/ˈraɪbəˌsoʊm, -boʊ-/^[1]) is a complex molecular machine, found within all living cells, that serves as the site of biological protein synthesis (translation). Ribosomes link amino acids together in the order specified by messenger RNA (mRNA) molecules. Ribosomes consist of two major components: the small ribosomal subunits, which read the RNA, and the large subunits, which join amino acids to form a polypeptide chain. Each subunit comprises one or more ribosomal RNA (rRNA) molecules and a variety of ribosomal proteins (r-protein or rProtein^[2][3][4]). The ribosomes and associated molecules are also known as the translational apparatus.

Overview

The sequence of DNA, which encodes the sequence of the amino acids in a protein, is copied into a messenger RNA chain. It may be copied many times into RNA chains. Ribosomes can bind to a messenger RNA chain and use its sequence for determining the correct sequence of amino acids. Amino acids are selected, collected, and carried to the ribosome by transfer RNA (tRNA) molecules, which enter one part of the ribosome and bind to the messenger RNA chain. It is during this binding that the correct translation of nucleic acid sequence to amino acid sequence occurs. For each coding triplet in the messenger RNA there is a distinct transfer RNA that matches and which carries the correct amino acid for that coding triplet. The attached amino acids are then linked together by another part of the ribosome. Once the protein is produced, it can then fold to produce a specific functional three-dimensional structure although during synthesis some proteins start folding into their correct form.

A ribosome is made from complexes of RNAs and proteins and is therefore a ribonucleoprotein. Each ribosome is divided into two subunits:

1. a smaller subunit which binds to a larger subunit and the mRNA pattern, and
2. a larger subunit which binds to the tRNA, the amino acids, and the smaller subunit.

When a ribosome finishes reading an mRNA molecule, these two subunits split apart. Ribosomes are ribozymes, because the catalytic peptidyl transferase activity that links amino acids together is performed by the ribosomal RNA. Ribosomes are often associated with the intracellular membranes that make up the rough endoplasmic reticulum.

Ribosomes from bacteria, archaea and eukaryotes in the three-domain system, resemble each other to a remarkable degree, evidence of a common origin. They differ in their size, sequence, structure, and the ratio of protein to RNA. The differences in structure allow some antibiotics to kill bacteria by inhibiting their ribosomes, while leaving human ribosomes unaffected. In bacteria and archaea, more than one ribosome may move along a single mRNA chain at one time, each "reading" its sequence and producing a corresponding protein molecule.

The mitochondrial ribosomes of eukaryotic cells, are produced from mitochondrial genes, and functionally resemble many features of those in bacteria, reflecting the likely evolutionary origin of mitochondria.^[5][6]

Discovery

Ribosomes were first observed in the mid-1950s by Romanian-American cell biologist George Emil Palade, using an electron microscope, as dense particles or granules.^[7] The term "ribosome" was proposed by scientist Richard B. Roberts in the end of 1950s:

During the course of the symposium a semantic difficulty became apparent. To some of the participants, "microsomes" mean the ribonucleoprotein particles of the microsome fraction contaminated by other protein and lipid material; to others, the microsomes consist of protein and lipid contaminated by particles. The phrase "microsomal particles" does not seem adequate, and "ribonucleoprotein particles of the microsome fraction" is much too awkward. During the meeting, the word "ribosome" was suggested, which has a very satisfactory name and a pleasant sound. The present confusion would be eliminated if "ribosome" were adopted to designate ribonucleoprotein particles in sizes ranging from 35 to 100S.

— Albert, Microsomal Particles and Protein Synthesis^[8]

Albert Claude, Christian de Duve, and George Emil Palade were jointly awarded the Nobel Prize in Physiology or Medicine, in 1974, for the discovery of the ribosome.^[9] The Nobel Prize in Chemistry 2009 was awarded to Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath for determining the detailed structure and mechanism of the ribosome.^[10]

Structure

Figure 2: Large (red) and small (blue) subunit fit together.

The ribosome is a highly complex cellular machine. It is largely made up of specialized RNA known as ribosomal RNA (rRNA) as well as dozens of distinct proteins (the exact number varies slightly between species). The ribosomal proteins and rRNAs are arranged into two distinct ribosomal pieces of different size, known generally as the large and small subunit of the ribosome. Ribosomes consist of two subunits that fit together (Figure 2) and work as one to translate the mRNA into a polypeptide chain during protein synthesis (Figure 1). Because they are formed from two subunits of non-equal size, they are slightly longer in the axis than in diameter.

Prokaryotic ribosomes are around 20 nm (200 Å) in diameter and are composed of 65% rRNA and 35% ribosomal proteins.^[11] Eukaryotic ribosomes are between 25 and 30 nm (250–300 Å) in diameter with an rRNA-to-protein ratio that is close to 1.^[12] Crystallographic work ^[13] has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis. This suggests that the protein components of ribosomes do not directly participate in peptide bond formation catalysis, but rather that these proteins act as a scaffold that may enhance the ability of rRNA to synthesize protein.

Figure 3: Atomic structure of the 30S subunit from Thermus thermophilus.^[14] Proteins are shown in blue and the single RNA chain in orange.

The ribosomal subunits of prokaryotes and eukaryotes are quite similar.^[15]

The unit of measurement used to describe the ribosomal subunits and the rRNA fragments is the Svedberg unit, a measure of the rate of sedimentation in centrifugation rather than size. This accounts for why fragment names do not add up: for example, prokaryotic 70S ribosomes are made of 50S and 30S subunits.

Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. Their small subunit has a 16S RNA subunit (consisting of 1540 nucleotides) bound to 21 proteins. The large subunit is composed of a 5S RNA subunit (120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 31 proteins.^[15]

prokaryotic ribosomes (E. coli)^[16]

ribosome	subunit	rRNAs	r-proteins
70S	50S	23S (2904 nt)	31
		5S (120 nt)
	30S	16S (1542 nt)	21

Affinity label for the tRNA binding sites on the E. coli ribosome allowed the identification of A and P site proteins most likely associated with the peptidyltransferase activity; labelled proteins are L27, L14, L15, L16, L2; at least L27 is located at the donor site, as shown by E. Collatz and A.P. Czernilofsky.^[17][18] Additional research has demonstrated that the S1 and S21 proteins, in association with the 3′-end of 16S ribosomal RNA, are involved in the initiation of translation.^[19]

Eukaryotes have 80S ribosomes, each consisting of a small (40S) and large (60S) subunit. Their 40S subunit has an 18S RNA (1900 nucleotides) and 33 proteins.^[20][21] The large subunit is composed of a 5S RNA (120 nucleotides), 28S RNA (4700 nucleotides), a 5.8S RNA (160 nucleotides) subunits and 46 proteins.^[15][20][22]

eukaryotic cytosolic ribosomes (R. norvegicus)^[23]

ribosome	subunit	rRNAs	r-proteins
80S	60S	28S (4718 nt)	49
		5.8S (160 nt)
		5S (120 nt)
	40S	18S (1874 nt)	33

During 1977, Czernilofsky published research that used affinity labeling to identify tRNA-binding sites on rat liver ribosomes. Several proteins, including L32/33, L36, L21, L23, L28/29 and L13 were implicated as being at or near the peptidyl transferase center.^[24]

The ribosomes found in chloroplasts and mitochondria of eukaryotes also consist of large and small subunits bound together with proteins into one 70S particle.^[15] These organelles are believed to be descendants of bacteria (see Endosymbiotic theory) and, as such, their ribosomes are similar to those of bacteria.^[15]

The various ribosomes share a core structure, which is quite similar despite the large differences in size. Much of the RNA is highly organized into various tertiary structural motifs, for example pseudoknots that exhibit coaxial stacking. The extra RNA in the larger ribosomes is in several long continuous insertions, such that they form loops out of the core structure without disrupting or changing it.^[15] All of the catalytic activity of the ribosome is carried out by the RNA; the proteins reside on the surface and seem to stabilize the structure.^[15]

The differences between the bacterial and eukaryotic ribosomes are exploited by pharmaceutical chemists to create antibiotics that can destroy a bacterial infection without harming the cells of the infected person. Due to the differences in their structures, the bacterial 70S ribosomes are vulnerable to these antibiotics while the eukaryotic 80S ribosomes are not.^[25] Even though mitochondria possess ribosomes similar to the bacterial ones, mitochondria are not affected by these antibiotics because they are surrounded by a double membrane that does not easily admit these antibiotics into the organelle.^[26]

High-resolution structure

Figure 4: Atomic structure of the 50S subunit from Haloarcula marismortui. Proteins are shown in blue and the two RNA chains in orange and yellow.^[27] The small patch of green in the center of the subunit is the active site.

The general molecular structure of the ribosome has been known since the early 1970s. In the early 2000s, the structure has been achieved at high resolutions, of the order of a few ångströms.

The first papers giving the structure of the ribosome at atomic resolution were published almost simultaneously in late 2000. The 50S (large prokaryotic) subunit was determined from the archaeon Haloarcula marismortui^[27] and the bacterium Deinococcus radiodurans,^[28] and the structure of the 30S subunit was determined from Thermus thermophilus.^[14] These structural studies were awarded the Nobel Prize in Chemistry in 2009. In May 2001 these coordinates were used to reconstruct the entire T. thermophilus 70S particle at 5.5 Å resolution.^[29]

Two papers were published in November 2005 with structures of the Escherichia coli 70S ribosome. The structures of a vacant ribosome were determined at 3.5 Å resolution using X-ray crystallography.^[30] Then, two weeks later, a structure based on cryo-electron microscopy was published,^[31] which depicts the ribosome at 11–15 Å resolution in the act of passing a newly synthesized protein strand into the protein-conducting channel.

The first atomic structures of the ribosome complexed with tRNA and mRNA molecules were solved by using X-ray crystallography by two groups independently, at 2.8 Å^[32] and at 3.7 Å.^[33] These structures allow one to see the details of interactions of the Thermus thermophilus ribosome with mRNA and with tRNAs bound at classical ribosomal sites. Interactions of the ribosome with long mRNAs containing Shine-Dalgarno sequences were visualized soon after that at 4.5–5.5 Å resolution.^[34]

In 2011, the first complete atomic structure of the eukaryotic 80S ribosome from the yeast Saccharomyces cerevisiae was obtained by crystallography.^[20] The model reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core. At the same time, the complete model of a eukaryotic 40S ribosomal structure in Tetrahymena thermophila was published and described the structure of the 40S subunit, as well as much about the 40S subunit's interaction with eIF1 during translation initiation.^[21] Similarly, the eukaryotic 60S subunit structure was also determined from Tetrahymena thermophila in complex with eIF6.^[22]

Function

Ribosomes are organelles that synthesize proteins. Proteins are needed for many cellular functions such as repairing damage or directing chemical processes. Ribosomes can be found floating within the cytoplasm or attached to the endoplasmic reticulum.

Translation

Ribosomes are the workplaces of protein biosynthesis, the process of translating mRNA into protein. The mRNA comprises a series of codons that dictate to the ribosome the sequence of the amino acids needed to make the protein. Using the mRNA as a template, the ribosome traverses each codon (3 nucleotides) of the mRNA, pairing it with the appropriate amino acid provided by an aminoacyl-tRNA. Aminoacyl-tRNA contains a complementary anticodon on one end and the appropriate amino acid on the other. For fast and accurate recognition of the appropriate tRNA, the ribosome utilizes large conformational changes (conformational proofreading) .^[35] The small ribosomal subunit, typically bound to an aminoacyl-tRNA containing the amino acid methionine, binds to an AUG codon on the mRNA and recruits the large ribosomal subunit. The ribosome contains three RNA binding sites, designated A, P and E. The A-site binds an aminoacyl-tRNA;^[36] the P-site binds a peptidyl-tRNA (a tRNA bound to the peptide being synthesized); and the E-site (exit) binds a free tRNA before it exits the ribosome. Protein synthesis begins at a start codon AUG near the 5' end of the mRNA. mRNA binds to the P site of the ribosome first. The ribosome is able to identify the start codon by use of the Shine-Dalgarno sequence of the mRNA in prokaryotes and Kozak box in eukaryotes.

Although catalysis of the peptide bond involves the C2 hydroxyl of RNA's P-site adenosine in a proton shuttle mechanism, other steps in protein synthesis (such as translocation) are caused by changes in protein conformations. Since their catalytic core is made of RNA, ribosomes are classified as "ribozymes,"^[37] and it is thought that they might be remnants of the RNA world.^[38]

Figure 5 : Translation of mRNA (1) by a ribosome

(2)(shown as small and large subunits) into a

polypeptide chain (3). The ribosome begins at th

 start codon of RNA (AUG) and ends at the stop

codon (UAG).

In Figure 5, both ribosomal subunits (small and large) assemble at the start codon (towards the 5' end of the RNA). The ribosome uses RNA that matches the current codon (triplet) on the mRNA to append an amino acid to the polypeptide chain. This is done for each triplet on the RNA, while the ribosome moves towards the 3' end of the mRNA. Usually in bacterial cells, several ribosomes are working parallel on a single RNA, forming what is called a polyribosome or polysome.

Addition of translation-independent amino acids

Presence of a ribosome quality control protein Rqc2 is associated with mRNA-independent protein elongation.^[39][40] This elongation is a result of ribosomal addition (via tRNAs brought by Rqc2) of CAT tails: ribosomes extend the C-terminus of a stalled protein with random, translation-independent sequences of alanines and threonines.^[41][42]

Würzburg University and Max Planck Institute researches, the results of which were published in Cell Reports and The EMBO magazines in September 2016, have shown that ribosomes have the role of being "a quality control point". Professor Utz Fischer from the University of Würzburg has been researching the assembly of proteins called "macromolecular machines" in the cell for years. He describes this assembly process as LEGO blocks: "Think of it as LEGO bricks at the molecular level: One brick is attached to the next until the product is finished. If only one defective or wrong brick is used, the entire building may be compromised as a result."^[43][44][45]

Ribosome locations

Ribosomes are classified as being either "free" or "membrane-bound".

Figure 6 : A ribosome translating a protein that is secreted into the endoplasmic reticulum.

Free and membrane-bound ribosomes differ only in their spatial distribution; they are identical in structure. Whether the ribosome exists in a free or membrane-bound state depends on the presence of an ER-targeting signal sequence on the protein being synthesized, so an individual ribosome might be membrane-bound when it is making one protein, but free in the cytosol when it makes another protein.

Ribosomes are sometimes referred to as organelles, but the use of the term organelle is often restricted to describing sub-cellular components that include a phospholipid membrane, which ribosomes, being entirely particulate, do not. For this reason, ribosomes may sometimes be described as "non-membranous organelles".

Free ribosomes

Free ribosomes can move about anywhere in the cytosol, but are excluded from the cell nucleus and other organelles. Proteins that are formed from free ribosomes are released into the cytosol and used within the cell. Since the cytosol contains high concentrations of glutathione and is, therefore, a reducing environment, proteins containing disulfide bonds, which are formed from oxidized cysteine residues, cannot be produced within it.

Membrane-bound ribosomes

When a ribosome begins to synthesize proteins that are needed in some organelles, the ribosome making this protein can become "membrane-bound". In eukaryotic cells this happens in a region of the endoplasmic reticulum (ER) called the "rough ER". The newly produced polypeptide chains are inserted directly into the ER by the ribosome undertaking vectorial synthesis and are then transported to their destinations, through the secretory pathway. Bound ribosomes usually produce proteins that are used within the plasma membrane or are expelled from the cell via exocytosis.^[46]

Biogenesis

In bacterial cells, ribosomes are synthesized in the cytoplasm through the transcription of multiple ribosome gene operons. In eukaryotes, the process takes place both in the cell cytoplasm and in the nucleolus, which is a region within the cell nucleus. The assembly process involves the coordinated function of over 200 proteins in the synthesis and processing of the four rRNAs, as well as assembly of those rRNAs with the ribosomal proteins.

Origin

The ribosome may have first originated in an RNA world, appearing as a self-replicating complex that only later evolved the ability to synthesize proteins when amino acids began to appear.^[47] Studies suggest that ancient ribosomes constructed solely of rRNA could have developed the ability to synthesize peptide bonds.^[48][49][50] In addition, evidence strongly points to ancient ribosomes as self-replicating complexes, where the rRNA in the ribosomes had informational, structural, and catalytic purposes because it could have coded for tRNAs and proteins needed for ribosomal self-replication.^[51] Hypothetical cellular organisms with self-replicating RNA but without DNA are called ribocytes (or ribocells).^[52][53]

As amino acids gradually appeared in the RNA world under prebiotic conditions,^[54][55] their interactions with catalytic RNA would increase both the range and efficiency of function of catalytic RNA molecules.^[47] Thus, the driving force for the evolution of the ribosome from an ancient self-replicating machine into its current form as a translational machine may have been the selective pressure to incorporate proteins into the ribosome’s self-replicating mechanisms, so as to increase its capacity for self-replication.^[51]

Specialized ribosomes

Heterogeneity in ribosome composition has been proposed to be involved in translational control of protein synthesis.^[56] Vincent Mauro and Gerald Edelman proposed the ribosome filter hypothesis to explain the regulatory functions of ribosomes. Emerging evidence has shown that specialized ribosomes specific to different cell populations can affect how genes are translated.^[57] Some ribosomal proteins exchange from the assembled complex with cytosolic copies ^[58] suggesting that the structure of the in vivo ribosome can be modified without synthesizing an entire new ribosome.