TRANSLATION

TRANSLATION

Translation means that genetic information copied into RNA with transcription is converted to a protein or polypeptide chain. In other words, it is the expression of genetic information in the form of protein molecules. Three important components of protein synthesis are messenger RNA (mRNA), transfer RNA (tRNA) and ribosomes. mRNA includes a code (password) which determines the protein’s amino acid sequence. The mRNA unit that encodes an amino acid and comprises three nucleotides is called codon. When the translation of mRNA starts, codons are read as successively without a space and hesitation. Each codon indicates either one aminoacid participating in protein synthesis or the termnation of protein synthesis. Code defines three nucleotide sequences called as codon and the

relationship between aminoacids .The 61 codons are codons encoding amino acids, they are also called sense codons . Degeneracy of the genetic code gives the meaning that aminoacids can be determined by more than one codon. Only tryptophan and methionine are determined y a single codon. Almost all of the aminoacids are determined by 2, 3 or 4 different codons, leucine, serin and arginine are determined by six different codons. The degenerate term is a term taken from the quantum physics by Francis Crick and it has homonymous to tell multiple physical condition. The genetic code is the same in almost all oganisms.

For example, codon indicates aminoacid arginine in AGA bacteria, in humans and other organisms. The universality of the genetic code takes place in strongest evidences where all living things share a common evolutionary heritage. Except few small exceptions, all viruses, prokaryotes, archaea, and eukaryotes use the same password dictionary. The codons which do not code any aminoacid are called

stop codons.

• UAG-amber codon

• UGA-opal codon

• UAA ochre codon 

Transfer RNA; it is a small, about 80 nucleotide RNA chain which adds specific amino acids to the growing polypeptide chain in protein synthesis in the ribosome during translation. 3 prime of tRNA has CCA nucleotide sequence and here is the region of amino acids connected . tRNA is an ‘adapter’ molecule which mediates the recognition of codon sequence in the mRNA, allows translation of codon to the suitable amino acid is hypothesized by Francis Crick. In Crick’s hypothesis of adapter molecule, while adapter molecule at one prime connects amino acids while the other prime it establishes a connection with amino acid-related mRNA sequence. In tRNA structure there is an anticodon region. On tRNA called as anticodon, there are nucleotide sequences of three bases which establish hydrogen

bonds with them to recognize codons in the mRNA. For example, tRNA anticodon nucleotide sequence which matches a mRNA strand as UUU shaped, is in the form of AAA. Anticodons are read in the direction of 3’->5’, in mRNA codons are read in the direction of 5’->3’. For example, if the anticodon base sequence is 3’-AAG5’, codon in mRNA is shaped as 5’-UUC-3’. If there was a specific tRNA for each amino acid codon in mRNA, there should be 61 kinds of tRNA.

Ribosomes, are found in all living cells and has primary importance in translation. About 65% of the ribosome is composed of rRNA, and 35% of the other part is composed of ribosomal proteins. They are cytosolic particles found in eukaryotic cells as 40S and 60S sedimantation coefficient of two subunits, a total of 80S sedimentation coefficient; and in prokaryotic cells, 30S and 50S sedimantation coefficient of two subunits, a total of 70S sedimentation coefficient. In 30S ribosomal subunit of prokaryotes, 16S ribosomal RNA is found. In 50S ribosomal subunit there are two types of rRNA, these are 5S and 23S rRNAs. In eukaryotes, in small ribosomal subunit 18S is found; in large subunit there are three types of

rRNA including 5S, 5.8S and 28S rRNAs. During the translation of Messenger RNA (mRNA) by ribosome, mRNA is located between these two subunits . During protein synthesis, many ribosomes on the same mRNA can form a polyribosome (polysome). Polysomes are observed in both eukaryotic and prokaryotic cells.  Polysomes in eukaryotic cells with 7-8 ribosomes per polysomes, are smaller than

polysomes in prokaryotic cells. A typical eukaryotic cell contains millions of ribosomes in its cytoplasm . A ribosome contains 4 binding sites. One of them is for mRNA and three of them are for tRNA (called the A-site, the P-site, and the Esite) A-Site (Acceptor Site): It is the site where specific tRNA anticodons linked with amino acids matches the bases located on the mRNA. P-Site (Peptidyl Site): It is the site where amino acids which are connected with a specific tRNA get transfer to the A-site are transferred to empty this site. E-Site (Exit Site): It is the site where tRNA separated from P-Site before leaving subunit, peptidyl site.

There are similar designs and functions of eukaryotic and prokaryotic ribosomes

Activating Enzymes

Aminoacyl-tRNA synthetase enzymes (M.A.: 40 000-100 000) are highly conserved proteins. Each of them containing ten enzymes is divided into two groups. One of them accesses with minor groove of tRNA acceptor helix, the other accesses with major groove of the same helix. Amino acids in cytoplasm are activated as aminoacyl-tRNA with binding to their specific tRNAs in the presence of ATP (adenosine triphosphate) with the help of activating enzymes called as aminoacyl-tRNA synthetases (aaRS) which are required Mg2+. A cell has 20 different aminoacyl-tRNA synthetases for each of 20 amino acids Amino-acyl tRNA synthetase enzyme use two step binds amino acids to tRNA molecule. In the first step, ATP and amino acid enter the reaction and amino acid with carboxyl group is connected to AMP (adenosine monophosphate), as a result, aminoacylAMP and pyrophosphate (PPi) are formed. In the second step, between amino acid and 3’OH group of adenosine ribose located in the 3 prime of tRNA molecule bind with water exit (ester bond) and AMP is separated. AMP is separated. In a conclusion, aminoacyl-tRNA is generated

1) Amino acid + ATP amino acyl-AMP+ PPi

2) Amino acyl-AMP + tRNA amino acyl-tRNA + AMP

Conclusion: Amino acid + ATP + tRNA amino acyl tRNA + AMP + PPi .

The translation can be divided into three stages:

Initiation

Elongation

Termination 

1) The Initiation of Translation

For translation, mRNA, tRNA ribosomes, as well as some protein factors are also required. In prokaryotes, for the initiation of translation requires IF1, IF2, and IF3 (initiation factors) protein factors. In eukaryotes, the number of protein factors are more than 12 (eIF1, eIF1A, elF2, eIF2B, eIF3, eIF4A, eIF4E, eIF4G, eIF5, eIF5B) and some of them play an important regulatory role. The initial stage consists of three basic step. Firstly, mRNA is bound to the ribosome small subunit. Secondly, by connecting the initiator tRNA to the mRNA, codon-anticodon match occurs. In the third stage, it is added to the initiation complex with the large subunit of the ribosome and protein synthesis proceeds .

Protein synthesis is started with AUG codon which is at the beginning of the mRNA-carried code. To distinguish initiation codon and other methionines, prokaryotes contain specific sequence Shine-Dalgarno sequence (UAAGGAGG) which is located about 5-10 nucleotides before the initiation codon. Close to the 3’ of 16S rRNA of 30S ribosomal subunit, there is a nucleotide sequence complementary to Shine-Dalgarno sequence, thus it accelerates the binding of mRNA to the 30S ribosomal subunit. In eukaryotes, the first codon located at the beginning of mRNA is 5’-AUG-3’ and it is recognized by specific initiation tRNA.

In prokaryotes, protein synthesis is initiated with N-formyl methionyl tRNA. In eukaryotes, protein synthesis is initiated with non-formylation of a special methionyl tRNA (tRNAi Met)

5‘- UAAGGAGG (5-10 base) AUG mRNA

3'--AUUCCUCC.........................16S rRNA

In prokaryotes, protein synthesis starts with the initiation complex. In prokaryotes when protein synthesis starts, the initial complex is formed in the presence of initiation factors (IF) and GTP near the 5’ of the mRNA mRNA molecule is binded to small 30S ribosome subunit. During initiation, initiation factor (IF-3) provides binding to the small subunit and prevent the binding to the large subunit. The second step is the binding of initiator fMet-tRNA to the initiation codon. In this step IF2 plays a role by binding tRNA and control entry of tRNA into the ribosome. IF2 in GTP-bound form is connected to 30S P site. After the binding of IF2, fMet-tRNA binds to IF2 and IF2 transfers fMet-tRNA to the P site. IF1 is associated with 30S ribosomal subunit in A site and prevents entry of aminoacyltRNA to the A site, increases the seperation of small and large subunits. It prevents the binding of initiator tRNA in small subunit to A site. 30S initiation complex is formed by 30S ribosome subunit, mRNA, initiator tRNA, GTP and initiation factors 

In the final stage of initation, 30S is divided from IF3, small subunit and 50S large subunit is joined to the initiation complex. In this conformation, the required energy is provided with by the hydrolyzes of guanosine triphosphate (GTP), into GDP and Pi by IF2 and IF1, and IF2 are separated. By the binding of the 50S large subunit, 70S initiation complex forms Eukaryotic mRNAs first interacts with ribosome with the 5’ cap structure; once ribosome recognize the cap structure, it moves to the 5’-AUG-3’ initiation codon. During movement, with the help of small subunit initiation factors, it finds the initiation codon in mRNA (AUG codon) with the base match with anticodon in initiator tRNA There are two other inducing translation of eukaryotic mRNA, one is in some mRNAs there is a purine three bases upstream of the initiation codon and a guanine downstairs (5’-G/ANNAUGG-3’) (Kozak sequence) It increases efficiency entering interaction with initiator tRNA

Kozak sequence:

5’–ACCAUGG–3’ Start codon

The second is poly-A tail of 3’ prime mRNA. It raises the translation level by supporting re-entering of ribosomes into the translation cycle. Shine-Dalgarno sequence is not available mRNA in eukaryotes unlike in prokaryotes IF1, IF2, IF3 factors and other additional proteins are replaced as equivalent initial factors in eukaryotes. elF3 is the initial factors as the equivalent of IF3 in prokaryotes. elF2 and elF5B, two proteins binding GTP help the binding of initiator tRNA. elf5B is the equivalent of IF2 in a dependent way of elF1A, the equivalent IF1, is connected to the small subunit; provides the binding of elf2 and initiator tRNA to the small subunit. The most important function of elF1A is to create 40 S preinitation complex by mediate the transfer of Met-tRNAf to the 40 S ribosomal subunits. Premise-initiation complex is formed (43S subunit, or the 40S and tRNA) elF4F enables recognizing of 43S preinitiation complex by mRNAs. Then, elF4B is bound to this complex. elF4B activates RNA helicase in elF4F. Helicase helps biding of 43S preinitiation complex to small subunit of mRNAs Correct base pairing of the small subunit and initiation factors triggers the separation of elF2 and elF3 during their movement along the mRNA and leads to the binding of large subunit to the small subunit. The connection of the large subunit results the separation of other initiation factors. Consequently, in the initial stage of translation in the eukaryotes 80S initiation complex is formed.

2) Elongation

The second stage in protein synthesis is the elongation stage, where amino acids are combined to form a polypeptide chain. It includes all occuring reactions from the first peptide bond formation to the last peptide bond formation in protein synthesis. 

In prokaryotes, to fulfill the elongation, the initiation cmplex, tRNAs charged with amino acids, elongation factors EF-Ts (elongation factor thermo stable), EF-Tu (elongation factor thermo unstable) and EF-G (historically known as translocase) and GTP are required . After the initial phase, ribosome bindly mRNA and fMettRNAfMet is positioned on the AUG initiation codon which is on the P site; A site is empty. Elongation occurs in three steps The first step is the binding of aminoacyl-tRNA matching the codon in mRNA at ribosome A site. After formation of the initiation complex, thanks to hydrolysis of GTP and elongation factor (EF-Tu) for the A site of this complex, aminoacyl-tRNA comes to anticodon to recognize the codons of mRNA. Before that, EF-Tu firstly is bound with GTP and then to create a three-ternary complex binds to a charged-tRNA. By binding to 3’prime of EF-Tu tRNA, it protect amino acid and peptide bond formation. The occuring three-ternary complex the ribosome A site and here mask it codon on the mRNA and anticodon on the tRNA match. EF-Tu interacts with factor binding site in the large subunit (GTPase activity triggered), hydrolyzes the particular GTP and

separates from EF-Tu–GDP complex and Pi ribosome released form of the Aminoacyl-Trna. EF-Tu-GDP is inactive and must be activated before the next elongation cycle. For this, EF-Ts is required, because affinity of EF-Tu for GDP is 40 times greater than its affinity for GTP. EF-Ts activates EF-Tu exchanging GDP with GTP. EF-Tu does not interact with fMet-tRNA because initiator fMet-tRNAis absent in the A site

EF-Tu·GTP + aminoacyl-tRNA EF-Tu·GTP- aminoacyl-tRNA

EF-Tu·GTP- aminoacyl-tRNA + ribosome ribozome·aminoacyl-tRNA +

EF-Tu·GDP +Pi

EF-Tu·GDP + EF-Ts EF-Tu·Ts + GDP

EF-Tu·Ts +GTP EF-Tu·GTP + EF-Ts

Reactions are similar in eukaryotic cells. Instead of elongation factors EF-Tu and EF-Ts in prokaryotes; there is a stable ternary complex, shows similar features as ’eEF1-alpha-beta-gamma’’. eEF1-alfa is eukaryotic equivalent of EF-Tu and eEF1-beta-gamma is eukaryotic equivalent of EF-Ts. The second step in the elongation is hydrolysis of ester bond of COOH group in the amino acid found in the P site and the formation of a peptide bond with NH2 group of the amino acid in A site. So, it is the transfer of extending polypeptide from tRNA in P site to amino acid of tRNA in A site. This reaction is called

peptidyl transferase reaction. For the formation of peptide bond in ribosome, the responsible region is peptidyl transferase center. The third step of the elongation phase is called as translocation. This step move positions ribosome on to the next codon and it is required the hydrolysis of elongation factor G(EF-G) and GTP into the GDP. After pepdidyl transferase reaction occurs, tRNA in P site breaks its bond with amino acid and extending polypeptide chain remains bound to tRNA in A site. For the addition of a new amino acid into the extending polypeptide chain, there is a EF-G’s role tRNA in A

site moves to P site. The mRNA shift is accomplished by base pairings between a moving tRNA in A site and mRNA. mRNA is taken with moving tRNA from A site. At exactly the same time, tRNA in P site moves to E (output) site and from here tRNA being freed in E site leaves ribosome. The movement of tRNA in P site to the E site breaks base pairings between tRNA and mRNA. EF-G factor is similar to EF-Tu but larger than it . EF-G binding to ribosome secures translocation. After peptidyl transfer reaction, with the separation of tRNA in A site, EF-G-GTP binds to A site. By hydrolysis of GTP, the three dimensional form of EF-G-GDP varies, triggers the translocation of tRNA from A site. When

translocation is completed, the affinity of ribosome with EF-G-GDP reduces and allows it to be released . Ribosomes can not interact with both EF-Tu and EF-G at the same time. These factors bind to ribosomes one by one. While the function of work one ends and leaves from complex, the other one is introduced to the ribozome. First, EF-Tu+GTP+amino acyl tRNA form ternary (triple) complex. After leaving of EF-Tu+GDP, EF-G+GTP is bound and then it is released as EFG+GDP Elongation in eukaryotic cells occurs in a similar manner. EF-G in prokaryotes is equivalent to eEf2 (Eukaryotic elongation factor 2) in eukaryotes. eEF2 promotes GTP-dependent ribosome translocation. This protein is completely inactivated with EF2 kinase phosphorylation. As a result of the translocation, the A site of the ribosome is empty and ribosome is ready to accept the next aminoacyl tRNA (aatRNA) and repeat the cycle. Until reaching a termination codon, this cycle continues

3)Termination

This step for release of the completed polypeptide includes necessary reactions. Aminoacyl tRNA binding in ribosome, formation of the peptide bond and translocation cycles continues until the arrival of one of the three termination codon (UAA, UAG, or UGA) to the A site. Because, there is no complementary tRNA of termination codon. When faced with a termination codon, no tRNA binds

to the A site of the ribosome. Instead, proteins called as release factors (RF) correspond to the termination codons and terminate the translation. When a termination codon came to A site of the ribosome, a RF recognizes this codon and binds to it 

Releasing factors are divided into two classes as Class 1 RFs and Class 2 RFs. In prokaryotes, there are two types of Class 1 RFs: RF1 and RF2. In eukaryotic cells, Class 1 RF is the only kind: eRF1. In prokaryotes, there are three releasing factor totally (RF1, RF2 and RF3). Release factor 1 is responsible for the recognition of termination codons UAA and UAG and Release factor 2 is responsible for the recognition of UAA or UGA. The other task of the RF1 and RF2 is to trigger polypeptide hydrolysis from tRNA in P site. RF1 and RF2 are similar in size and shape with tRNA and bind A site of the ribosome as it does during the elongation cycle of amino acid-tRNA-EF-Tu-GTP complex. Release factor 3 forms complex with GTP and binds ribosome. Release factor 3 structurally is similar to EF-G . Then release factor promotes the bound of tRNA in P site from polypeptide chain, the link between the last amino acid in polypeptide chain and tRNA is then broken. In this process, RF3 bound GTP is hydrolized to GDP. With the help of release factors the last released tRNA molecule and polypeptide chain are separated from ribosome; the large subunit of the ribosome is separated from mRNA and the small subunit The termination of protein synthesis in eukaryotic cells is performed in a similar manner as of prokaryotes. However, 2 different release factors are located. The first one; eRF1; recognizes all three termination codons . It can be said that it is the equivalent of RF1 and RF2 in prokaryotes. eRF3 binds with GTP and stimulates the separation of polypeptide from ribosome, with similar tasks with RF3 in prokaryotes. By providing termination, release factors enable protein synthesis cycle to end.

Ribosome Recycling

After release of polypeptide chain and RFs, ribosome (in its P and E site) is still connected to mRNA and uncharged tRNA. For ribosome enter a new cycle of polypeptide synthesis, it needs to release the mRNA and tRNA, to separate thearge and small subunits of the ribosome. All of these events are called as the reuse of ribosome . In prokaryotes, the activating of ribosome recyling after the release of polypeptide for recycling ribosomeis involves EF-G and IF3. RRF (ribosome recyling factor) binds to A site of the ribosome which is empty and imitates a tRNA; at the same time it allows the binding of EF-G to the ribosome; stimulates the release of uncharged tRNAs in P and E sites. IF3 (being initiation

factor) leads release of mRNA and separation of the two subunits of the ribosome. These subunits can participate in a new cycle.

A COMPARISON OF PROKARYOTIC AND EUKARYOTIC TRANSLATION

In bacterial and eukaryotic cells, along the process of translation major similarities and differences are seen First of all, although prokaryotes and eukaryotes have similar genetic codes, amino acid identified by initiation codon is different. In bacteria, the modified form of methionine, N-formyl methionine (fMet) is the first amino acid which enters the structure of whole polypeptide and encrypted with 5’- AUG -3’ codon. In eukaryotes, methionine is not formylated. Other difference is the existence at the same time of transcription and translation in bacterial cells. But in eukaryotes there is a nuclear membrane, so that, transcription and translation are separated from each other. The physical separation of transcription and translation is important for the control of gene expression. Another difference is that in eukaryotic cells, mRNA life (hours and days) is more long than mRNA life in prokaryotes (a few minutes). After transcription ended in eukaryotic cells, protein synthesis can persist for a long time, but it ends up very fast in prokaryotic cells. In both bacterial and eukaryotic cells, aminoacyl-tRNA synthetases conjugate to amio acids to their cognate tRNAs. Chemical reactions are the same. The differences include in the size and binding of bacterial and eukaryotic ribosomal subunits. For example, the large subunit of eukaryotic ribosome contains three rRNAs, but bacterial ribosome contains only two rRNA come for each subunit.

In the process of initiation, the number of initiation factors involved in eukaryotic cells is more than the number of initiation factors involved in prokaryotic cells. Also, in prokaryotic cells there is a Shine- Dalgarno consensus sequence while eukaryotic cells has a different ribosome binding sequence as Kozak sequence. Although elongation and termination process in prokaryotic and eukaryotic cells

are similar, different elongation factors and release factors are used.