The processed RNA molecules take part in protein synthesis with the help of ribosomes.
All the three types of RNAs are involved in protein synthesis in the following main steps:
(i) Activation of amino acids,
(ii) Transfer of amino acid to tRNA,
(iii) Initiation of protein synthesis,
(iv) Elongation of the polypeptide chain.
During the process of translation the genetic information’s are coded in mRNA transcripts in the form of codons which in turn are specifically read by anticodon of tRNA and used to form a polypeptide molecule of defined function.
1. Charging of tRNA:
(i) Activation of Amino Acids:
In protein synthesis only L-amino acids take part. The D- amino acids are screened from the all 20 amino acids. In addition, the other amino acids which are not used in protein synthesis are citrulline, alanine, β-alanine, etc. Each amino acid has a specific aminoacyl tRNA synthetase (charging enzyme) and a specific tRNA. At least 32 tRNAs are required to recognise all the amino acid codons, but some cells used more than 32.
However, these may be more than one species of tRNA for a specific amino acid but there is only one charging enzyme for each amino acid. Its carboxyl group activates the amino acids through being catalysed by its own specific activating enzyme (aminoacyl tRNA synthetase) in the presence of ATP. Consequently aminoacyl AMP synthetase complex is formed which remains in bound form with the activating enzyme.
(ii) Transfer of Amino Acid to tRNA:
The process of transfer of activated amino acids to tRNA is called charging of tRNA. The tRNAs are specific to their specific amino acid. Therefore, tRNAs are named according to specific amino acid such as tRNAala (for alanine), tRNAval (for valine), etc. Therefore, the activated amino acid is transferred to its specific tRNA.
The aminoacyl- AMP-synthetase complex formed as above is transferred to tRNA as below:
Aminoacyl-AMP- synthetase complex + tRNA → Aminoacyl – tRNA + AMP + aminoacyl tRNA synthetase Structure of aminoacyl tRNA is given in Fig. 10.14. The aminoacyl-AMP synthetase reacts with specific tRNA and forms aminoacyl-tRNA complex by releasing the enzyme aminoacyl- tRNA synthetase.
This shows that the enzyme tRNA synthetase has two specific sites. One site recognises the specific amino acids and the other site recognises the specific tRNA molecule. Thus, the tRNA synthetase brings the specific amino acid and tRNA molecule together.
However, these recognition properties are essential for making sure that the specific amino acid is charged on the proper tRNA molecule. In the same way the tRNA molecule also consists of two specific sites, one site for recognising its specific aminoacyl- tRNA synthetase and the second (the anticodon) for codon present on mRNA molecule.
For the incorporation of an amino acid at proper position in the polypeptide chain, recognition of codon on mRNA by the specific anticodon on tRNA is required.
2. Initiation of Polypeptide Synthesis:
There are several specific and complex processes (Fig. 10.15) that are involved in the initiation and continuation of the elongation of polypeptide sequence. The essential components required for initiation are: initiation factors, ribosome, mRNA, guanosine triphosphate (GTP) and aminoacyl-tRNase.
Fig. 10.15 : Initiation of translation.
(i) Initiation Factors:
There are certain initiation factors (IF) which are required for the initiation of protein synthesis. In prokaryotes three IF (i.e.IF-1, 9,000 MW; IF-2, 1,15,000 MW and IF-3,22,000 MW) are involved in the initiation process, whereas in eukaryotes no IF equivalent to IF-1 and IF-2 are found.
However, IF-2 is functionally equivalent to eukaryotic eIF-2 and elF- 2′ and IF-3 is equivalent to eukaryotic eIF-3. The IF-1, IF-2 and IF-3 are present in the 30S subunit of the ribosome. IF-1 and IF-2 help in binding of initiation tRNA (tRNAmet) to the 30S ribosome subunit.
(ii) Formylation of Methionine:
Methionine is the starting N-terminal amino acid in eukaryotes, whereas in prokaryotes methionine consists of a formyl group (-CHO). Therefore, formyl group containing methionine is called N-formyl methionine. In prokaryotes as well as in eukaryotes initiation of protein synthesis occurs through a specific methionyl tRNA which is commonly known as initiation tRNA (i.e. tRNAmet).
Binding of initiation tRNA with methionine/formylmethionine occurs as below:
Methionine + tRNA → Methionine – tRNA (met – tRNA)
Formyl tetrahydrofolate + NH2-methionyl tRNA Transformylase → N-formyl-methionyl-tRNA (N-fmet-tRNA)
(iii) Formation of 30S Initiation Complex:
The first step in initiation of protein synthesis is the formation of 30S initiation complex. This complex consists of an mRNA, 30S ribosomal subunit, GTP, IF (1, 2 and 3) and the initiator tRNA i.e. N-fmet-RNA.
Formation of 30S initiator occurs in the following steps (the actual order of these steps is not known):
(a) The initiation factors (IF-1, IF-2 and IF-3) bind to 30S ribosomal subunit in the presence of GTP to form 30S-IF complex (Fig. 10.15A). However, when the mRNA is absent IF-1 and IF- 3 do not form complex neither with 30S subunit nor 50S subunit.
(b) The second step involves the association of mRNA and initiator tRNA to the 30S subunit. However, the actual order of these steps vary. The IF-3 can bind to both 30S subunit and to mRNA. The 30S-IF complex binds to mRNA at the site containing initiation codon (in the order of pB reference AUG, GUG, UUG, CUG, AUA or AUU). Each mRNA at its un-translational region consists of a ribosome binding site for every polypeptide in the form of polycistronic message.
This ribosome binding site (i.e. 5′-AGGAGGU-3′) is known as Shine-Dalgarno sequence which is important in the binding of mRNA to the 30S-IF complex (Fig. 10.15B). The Shine-Dalgarno sequence base pairs to a region present at 3′ end of 16S rRNA. This pairing will results in proper position of initiating AUG codon so that it can combine with an initiator anticodon on tRNA.
(c) The IF-2 which has combined with GTP, permits the initiator tRNA (i.e.N-fmet-tRNA) to bind to the 30S ribosomal subunit (Fig. 10.15C). Then it allows to the 30S and 508 subunits to get associated. This binding is followed by removal of IF-3 from the 30S-IF complex.
Removal of IF-3 is necessary because its presence inhibits the association of two ribosomal subunits. At this stage the initiation complex consists of mRNA associated with the 30S ribosomal subunits, IF-1, IF-2-GTP and fmet-tRNA.
(iv) Formation of the Complete Initiation Complex:
The last step in prokaryotes is the union of the 30S initiation complex with 50S ribosomal subunits and formation of a complete 708 initiation complex (Fig. 10.15D). This process of union causes the immediate hydrolysis of the bound GTP to GDP + Pi.
The process of union is accomplished in the presence of an analogue of GTP (i.e. 5’guanyl methylenediphosphate). Therefore, hydrolysis of GTP and subsequent removal of GDP is essential for the IF-1 dependent release of IF-2 from the ribosome (Fig. 10.15 E). Similarly, in eukaryotes the 40S initiation complex is attached to 60S ribosomal subunit and forms the complete 80S initiation complex.
The ribosome has three important binding sites, two are important in protein synthesis. The two binding sites are: the aminoacyl-tRNA binding site (A), the peptide (P) binding site and the E site (Fig. 10.15E).
The A site receives all the incoming charged tRNA, whereas the P site possesses the previous tRNA with the new polypeptide (peptidyl tRNA) attached. The fmet-tRNA (initiation tRNA) directly binds with P site, but not A site. Function of the E site is de-acylation.
3. Elongation of Polypeptide Chain:
As shown in Fig. 10.15 E, at the end of initiation sequence, the 70S ribosome possesses the fmet-tRNA in the P site, whereas the A site is free to receive the next aminoacyl-tRNA according to the codons on mRNA. The addition of amino acids to the growing polypeptide chain as per codon on mRNA is called elongation of chain.
The rate of addition of amino acid to the growing polypeptide is about 16 residues per second at 37°C. The 5S rRNA molecule recognises the nucleotide sequence of TѰ loop of tRNA and thus helps in binding of tRNA to the A site. The codons direct the specific aminoacyl-tRNA to form bonds. The bond formation is stimulated by an elongation factor T (EF- T) and GTP. T refers to transferase activity.
The elongation factor (EF) is a soluble protein which is required for elongation of polypeptide chain. The EF is of two types, EF-T and EF-G. The EF-T is associated with transferase activity, whereas the EF-G is involved in translocation of mRNA.
In prokaryotes the EF-T consists of two protein subunits which are called EF-Tu (temperature unstable, MW 44,000) and EF-Ts (temperature stable, MW 30,000). The EF-Tu is most abundant protein in E.coli that accounts for 5-10% of the total cellular protein. Both the proteins (EF-Tu and EF-Ts) are needed for binding the aminoacyl- tRNA to the ribosome.
The eukaryotic EF is called EF-1 and EF-2 which has resemblance with the prokaryotic EF-T and EF-G. More specifically the EF-1 is like the EF-Tu in its structure and function. At a time the EF-1 exists in one of the two forms (light form, EF- 1L and heavy form, EF- 1H).
The function of EF-2 is translocation of aminoacyl-tRNA from A site to the P site. The GTP is required to drive the process of chain elongation. Bermerk (1978) has discussed the mechanism of chain elongation on ribosome.
Elongation of the polypeptide chain is accomplished in the following two steps:
Fig. 10.16 : Events of polypeptide chain formation.
(i) Binding of Aminoacyl-tRNA to the A Site:
The GTP binds to EF-T and splits it into EF- Tu-GTP and EF-Ts. The EF-Tu-GTP can bind to all aminoacyl-tRNA (except the initiator tRNA) and results in formation of GTP-EF-Tu-aminoacyl-tRNA complex (Fig. 10.16A). It is an intermediate complex which is bound to the ribosome.
In this step the EF-Ts complex does not play any role. After the aminoacyl-tRNA binds to the A site, GTP is hydrolysed and EF-Tu-GDP complex is released from the ribosome (B). Each aminoacyl -tRNA bound hydrolyses one GDP. The aminoacyl – tRNA may bind to the A site but this binding may not be followed by EF-Tu release from the ribosome. This shows that the purpose of GTP hydrolysis is the release of EF-Tu from the ribosome.
(ii) Peptide-Bond Formation:
Soon the enzyme peptidyl transferase (PTas) catalyses the peptide-bond formation. In fact this is catalysed by the 23S rRNA. This process is called peptidyl transfer (Fig. 10.16.C).
However, peptide bond formation depends on release of EF-Tu from the ribosome but not on hydrolysis of GTP. The EF-Ts complex recycles the EF-Tu-GDP to EF-Tu- GTP, but does not cause release of EF-Tu from the ribosome as the release of IF-2 depends on IF-1.
When a new aminoacyl-tRNA binds to the A site, peptide bond formation occurs between the starting amino acid (N-fmet-tRNA on prokaryotes and met-tRNA in eukaryotes) and new aminoacyl-tRNA at the P site.
The enzyme peptidyl transferase located in 50S ribosomal subunit catalyses the formation of peptide bond between the amino group of new incoming amino acid and the C-terminal of the elongating polypeptide attached to tRNA (Fig. 10.16D). During the process of bond formation, H2O is eliminated.
When the peptide bond is formed, the growing peptide chain binds to the tRNA that carries the incoming amino acid and occupies the A site of ribosome. The discharged tRNA after dissociating itself from the peptide chain is released from the P site (Fig. 10.16D-E). It is known so far that ribosome consists of two sites (A and P) but the recent evidences suggest that it consists of three sites: A, P and E. The E site is specific for de-acylated tRNA (E).
(i) Mechanism of Translocation:
In the ribosome at site A (aminoacyl-tRNA accepting site) the incoming aminoacyl-tRNA enters where decoding (codon-anticodon recognition) takes place. Thereafter, the ribosome moves along mRNA and, therefore, a change in complex occurs.
The movement of ribosome causes the alignment with A site of next codon of mRNA to be translated. Consequently, the peptidyl-tRNA situated at A site is transferred to P site. This event of transfer of peptidyl – tRNA is called translocation (Fig. 10.16E-F).
During translocation the events that are accomplished are:
(i) Removal of discharged tRNA from the P site,
(ii) Movement of the peptidyl tRNA from the A site to the P site, and
(iii) Movement of message by one codon.
The recent model of ribosome shows that:
(i) The incoming charged tRNA binds at the A site,
(ii) The growing polypeptide attached to tRNA and bound to P site is transferred to the tRNA in the A site, and
(iii) The newly deacaylated tRNA after translocation is not released immediately but are bound to the E site.
Now both the E and P sites are engaged. The other incoming charged tRNA binds to the unoccupied A site. This causes reduction in affinity of the E site for the deacylated tRNA and resulting in release of discharged tRNA from the ribosome. The process of binding of incoming aminoacyl-tRNA to site A continue until the termination signal is received (G-I).
In prokaryotes translocation is brought by the EF-G or translocase (MW, 80,000 in which G = GTPase) and GTP hydrolysis are required. EF-G binds to the same site as the EF-Tu. After binding EF-G hydrolyse the ATP to ADP + Pi in the presence of ribosome.
It is obvious that during elongation two molecules of GTP are hydrolysed per peptide bond, one is EF-T dependent and the other EF-G dependent. The EF-G is released from the ribosome after each step of elongation. Since both EF-T and EF-G utilize the same binding site, elongation cannot continue unless EF-G is released.
5. Termination of Polypeptide Chain:
(i) Recognition of Termination Signal:
The polypeptide chain continues the elongating until a termination codon on mRNA reaches to ribosome. The termination codons (UAA-ochre, UAG- amber, UGA-opal or umber) are also called as non-sense codon because no tRNA anticodon pairs with them. It is not necessary that the termination codon is the last codon of mRNA.
For example in bacteria and bacteriophages polygenic mRNAs are common and they consist of a number of initiation and termination codons. After translocation of one of the above termination codons into the A site, the ribosome does not bind to an aminoacyl-tRNA-EF-Tu-GTP complex. Then it receives the signal of termination.
(ii) Release of Polypeptide Chain:
When a termination codon is translocated into the A site, the ribosome instead of binding with a complex containing an amino acid, binds with a peptide release factor (RE) (Fig. 10.16 J). In prokaryotes there are three RF proteins (RF-1, MW 44,000; RF-2, MW 47,000; RF-3, MW 46,000).
The RF-1 is active with UAA and UAG codons and the RF-2 is active with UAA and UGA codons. The RF-3 activates the RF-1 and RF-2; therefore, the RF-3 is called stimulatory (S) factor. In eukaryotes there is only one RF protein (MW 56,500 and 1,15,000) which is active with codons UAA, UAG and UGA. The RF protein exists in two units and both of them remain in active form.
The ribosome binds either with RF-1 or RF-2. However, the RF protein activates peptidyl transferase which hydrolyses the bond joining the peptide to the tRNA at the site P. This results in release of the peptide chain (Fig. 10.16J).
6. Post Translational Processing:
After release some of the processing events occur in the polypeptide chain.
Such modifications occur both in prokaryotes and eukaryotes as given below:
(i) Removal of fmet from the Polypeptide Chain:
The formyl group of the N-terminal fmet is removed by the enzyme methionine deformylase. The enzyme formylmethionine specific peptidase (methionyl amino-peptidase or MAP) hydrolyses the entire formylmethionine residues. All the terminal methionine’s are not removed because there is involvement of discrimination in channeling of different polypeptides through these two alternative steps.
The side-chain penultimate amino acid acts as the discriminating factor. Removal of methionine by MAP depends on the length of side chain. The side chain of the longer length has less possibility for MAP to remove the methionine. The other processing’s are acetylation (of L12 to give rise L7) or adenylation.
(a) Loss of signal sequences:
In some polypeptides, about 15 to 30 amino acid residues are present at N-terminus. These residues act as signal sequence and direct the protein to its ultimate destination. The signal sequences are cleaved by specific peptidases.
(b) Modification of individual amino acid:
Some amino acid side chains are specifically modified such as:
(a) Enzymatic phosphorylation by ATP of -OH group of certain amino acids (e.g. serine, threonine, tyrosine),
(b) Binding of Ca++ to phosphoresine groups of milk protein, casein,
(c) Addition of carboxyl (-COOH) group to aspartate glutamate residues of some proteins (e.g. blood clotting protein, prothrombin),
(d) Methylation of proteins (e.g. methylation of lysine residues in cytochrome c, calmodulin).
(c) Formation of disulphide cross-links:
Disulphide bridges between cysteine residues of some proteins are formed. Hence, they are covalently cross-linked and attain native from.
Attachment of the carbohydrate side chains during or after protein synthesis is called glycosylation, for example glycoproteins.
(e) Addition of prosthetic group:
Prosthetic groups get covalently bound to many prokaryotic and eukaryotic proteins. For example, biotin molecule is covalently linked to acetyl-CoA carboxylase.
(ii) Ribosome Editing:
During the process of translation certain inappropriate amino-acylated – tRNAs enter the A site of ribosome and remain bound to the ribosome. These outnumber the appropriate amino acylated (aa)-tRNAs.
However, the aa-tRNAs remain bound for a long time to the A site for a peptide from P site of ribosome. There are two processes that can reduce the error of surviving polypeptide chain e.g. ribosome editing and preferential degradation of polypeptide chain containing erroneous amino acids.
According to the ribosome editing hypothesis the structure of inappropriate peptidyl-tRNA does not correctly complement the structure of mRNA; therefore, it dissociates from the ribosome during protein synthesis. However, the gene relA produces a signal molecule (alarmone), guanosine tetra-phosphate (ppGpp) which affects the editing process.
The ppGpp interacts with EF-G and results in longer life of peptidyl-tRNA in the A site and en-chances the editing process. The gene pth synthesizes peptidyl-tRNA hydrolase that acts upon the peptidyl-tRNA when it is released. The ribosome free peptidyl-tRNA is hydrolysed by this enzyme. Consequently an intact peptide and an intact tRNA are produced. The defective peptide is degraded by the enzyme.
(iii) Protein Folding:
After the synthesis of polypeptide chain, it undergoes spontaneous foldings. The secondary folds are formed between the folded regions. Finally as a result of further folding there develops a tertiary structure of polypeptide chain i.e. protein. Before the terminal amino acids are added in polypeptide chain, the protein reaches to its final shape during the course of chain termination.
According to the recent theories the process of protein folding is complex. Many proteins require assistance to get folded properly. This assistance is provided by the proteins of special kind known as chaperones or chaperonins. These assist polypeptides to self assemble by inhibiting alternative assembling pathway.
When chaperones interact with the polypeptide, the chance of incorrect in-folding is reduced. In E. coli the example of chaperones are GroE1 (60 KDa), GroES (10KDa) and DnaK (70KDa). All these are constitutive proteins which increase their concentration when there is stress like heat shock.
7. The Signal Hypothesis (Protein Export):
It is interesting to note that after synthesis of protein, how it is incorporated in membranes or secreted outside by the cell? However, it is believed that the secretory proteins are synthesised by the ribosomes which are attached to the endoplasmic reticulum and released into it. From endoplasmic reticulum they are transported to various cell organelles (in eukaryotes) where from secreted outside the cell through the process of exocytosis.
To explain this mechanism Blobel and Dobberstein (1975) proposed a theory known as signal hypothesis. Furthermore Blobel (1978) reviewed this hypothesis and postulated that the mRNAs that translate secretory proteins possess on 3′ side of initiation codon (AUG) a group of signal codons.
The endoplasmic reticulum consists of ribosome receptor proteins. A polypeptide chain consisting of a special region (signal peptide region) is synthesized by the ribosome. After coming out from the ribosome the signal peptide interacts with ribosome receptor protein and results in formation of a tunnel in the membrane.
However, the membrane tunnel coincides with the ribosomal tunnel. The enzyme signal peptidase breaks the polypeptide chain which is being synthesised. Upon complete synthesis of polypeptide chain, it is released inside the space of endoplasmic reticulum.
At the end the ribosomes dissociate from the membrane of endoplasmic reticulum; ribosome receptor proteins get diffused and close the tunnels. This process of entering the proteins into the membranes is also known as protein export.
A significant work has been done in recent years on secretory protein translocation system in E. coli and the other bacteria as well such as Bacillus subtilis. Salmonella typhimurium, Pseudomonas fluorescens, Enterobacter aero genes. Vibrio cholerae, Klebsiella oxycota, etc.
Most of the secretory proteins are translated first in a form of precursor containing 15-30 amino acids at N-terminus which is called a signal sequence. The signal sequence consists of a hydrophobic region of about 11 amino acid residues and a short stretch of hydrophilic region at N-terminus. The signal sequence is involved to bind the nascent polypeptide to the membrane.
In Gram-negative organisms the pathway for export and secretion of signal sequence-containing proteins is called general secretory pathway. The first step is the sec gene product dependent translocation of exported protein outside the cytoplasmic membrane. Brondage (1990) have presented a model of proOmpA (an export protein) translocation across the plasma membrane (Fig. 10.17).
Fig. 10.17 : A model for translocation of proOmpA across the plasma membrane.
The protein SecB (a product of secB gene) is a pilot chaperonin. It is associated with the protein that is to be transported i.e. transport protein, for example proOmpA. It is also synthesised on the ribosome. However, in the absence of SecB, the proOmpA aggregates and checks its insertion into the membrane.
The protein SecA is basically an ATPase and also forms a part of pre-protein translocase in association with integral membrane protein SecY/E. The SecA protein acts as a receptor for proOmpA-SecB complex. Subsequently, the ATP is hydrolysed releasing the proOmpA into the membrane. It also drives the overall chaperones and membrane-associated reactions.
Once the process of transport has been started at the expense of ATP, further translocation event proceeds through a series of trans-membrane intermediates, the energy requirement of which is met by proton motive force rather than ATP hydrolysis. During the process of translocation, the enzyme signal peptidase (LepB) cleaves the signal sequence of the exported protein.
It has to enter the periplasm or translocate across the outer membrane. Pugsley (1993) has published the complete general secretary pathway in Gram-negative bacteria where the various branches direct proteins to their final extra cytoplasmic destination (Fig. 10.18).
Fig. 10.18 : Main branches of the general secretory pathways (GSP) of Gram-negative bacteria. IMP, integrated membrane proteins; CAC, chaperone assembly channel; PSI, periplasmic secrection intermediates; SP, signal peptidase.
8. The Inhibitors of Gene Expression:
There are several antimicrobial agents that inhibit protein synthesis at two steps, either during transcription or translation. Franklin and Snow (1991) have nicely discussed the biochemistry of the action of antimicrobial antibiotics.
(i) Inhibitors of Transcription:
The rifamycin and chemically similar group (streptovarcins) inhibit the initiation of transcription. Rifampin and streptovarcin are the semisynthetic compounds, whereas rifamycins are the naturally occurring antibiotics. They tightly bind to P subunit of RNA polymerase (rpoB) and inhibit initiation of transcription.
It is similar to rifamycin because it too binds with P subunit of RNA polymerase. In addition, it inhibits both the processes: chain initiation and chain elongation in vitro.
(ii) Inhibitors of Translation:
It inhibits the activity of peptidyl transferase after binding to 508 subunit of bacterial ribosome. Its effect is bacteriostatic i.e. after removal of drug the effect is soon reversed. However, its effect in eukaryotes is the same as in prokaryotes. Bone marrow toxicity results in aplastic anaemia.
This group of antibiotic shows broad spectrum bacteriostatic activities against Gram-positive and negative bacteria, mycoplasmas, rickettsiae and chlamydiae. These prevent the binding of aminoacyl -tRNA to the A site of 30S ribosome. Moreover, it can bind to several sites of both 30S and 50S subunits.
(c) Cycloheximide (actidione):
It inhibits protein synthesis in eukaryotes (e.g. yeasts, fungi, higher plants and mammals) but not the prokaryotic microorganisms. It interferes with the activity of ribosome present in cytoplasm but not in mitochondria, by binding with 80S subunits and preventing the movement of mRNA.
This is a large group of antimicrobial agents that includes erythromycin, leucomycin, macrocin, carbomycin, chalcomycin, angalomycin, etc. These are active against the Gram-positive bacteria and less active against Gram-negative bacteria but not against the eukaryotes. These interact with SOS subunit of ribosome and inhibit protein synthesis. Also, they stimulate the dissociation of peptidyl-tRNA from the ribosome through abortive translocation step.
Lincomycin and the other chemically similar antibiotics inhibit peptidyl transferase through binding to 23S rRNA of the 50S subunit. These affect both Gram-positive and Gram-negative bacteria.
It binds to a peptide with the C-terminus of growing polypeptide and results in premature termination of polypeptide chain. It interacts with the P site of ribosome but not A site. It works equally well on 70S and 50S ribosomes.