In this essay we will discuss about Protein Synthesis. After reading this essay you will learn about: 1. Mechanism of Protein Synthesis 2. Ribosomes in Protein Synthesis 3. Non-Ribosomal Polypeptide Synthesis 4. Transfer RNA (tRNA) 5. Activation and Formation of Aminoacyl tRNA Molecules 6. Initiation 7. Energetics.
Contents:
- Essay on the Mechanism of Protein Synthesis
- Essay on the Ribosomes in Protein Synthesis
- Essay on the Non-Ribosomal Polypeptide Synthesis
- Essay on the Transfer RNA (tRna) in Protein Synthesis
- Essay on the Activation and Formation of Aminoacyl tRNA Molecules in Protein Synthesis
- Essay on the Initiation of Protein Synthesis
- Essay on the Energetics of Protein Synthesis
Contents
- Essay # 1. Mechanism of Protein Synthesis:
- Essay # 2. Ribosomes in Protein Synthesis:
- Essay # 3. Non-Ribosomal Polypeptide Synthesis:
- Essay # 4. Transfer RNA (tRna) in Protein Synthesis:
- Essay # 5. Activation and Formation of Aminoacyl tRNA Molecules in Protein Synthesis:
- Essay # 6. Initiation of Protein Synthesis:
- Essay # 7. Energetics of Protein Synthesis:
Essay # 1. Mechanism of Protein Synthesis:
Proteins are complex macromolecules consisting of a series of polypeptide chains made up of varying amounts of 20 different amino acids.
In addition to their primary sequence, protein chains have secondary and tertiary structures that localize hydrophilic (water-loving) and hydrophobic (water-hating) areas. Protein biosynthesis is an extremely important mechanism because the functions served by the proteins are numerous, ranging from catalysis of cellular reactions to participation in structural cellular elements.
Large amounts of proteins may remain stored in some plant tissues as a source of energy and nitrogen to be utilised during periods of rapid growth. Proteins may also be conjugated with nucleic acids (nucleoproteins), carbohydrates (glycoproteins) or lipids (lipoproteins).
Crick in 1958 enunciated a rule that has continued to be known as the central dogma of molecular biology. Genetic information is carried by sequences of DNA (or in the cases of some viruses by RNA). Through the stages of transcription and translation, they may be converted into the amino acid sequence of protein.
The situation described by the central dogma (Fig. 10.18) is:
The mechanism of protein synthesis involves process of translation because the language of the four base-pairing letters of nucleic acids is translated into the language of twenty letters of proteins. Translation is a complex process and in fact it necessitates the coordinated interplay of more than a hundred kinds of macromolecules.
Through the process, a protein is synthesized in amino-to-carboxyl direction by sequential addition of amino acids to the carboxyl end of the growing peptide chain. The activated precursors are amino acyl tRNAs, in which the carboxyl group of an amino acid is the 3′ terminus of a tRNA. The covalent linking of an amino acid to its corresponding tRNA is catalysed by a specific aminoacyl tRNA synthetase.
A current, generalized picture of protein synthesis would require the following participants:
1. A pool containing adequate amount of 20 amino acids commonly found in proteins.
2. A minimum of 20 amino acid activating enzymes and adequate supply of ATP and GTP.
3. A pool of more than 20 but fewer than 64 (= 43, the possible number of triplet codons) tRNA molecules are required, as there is more than one codon (degenerate codons) specifying a single amino acid in some cases.
4. A pool of subcellular ribonucleoprotein particles called ribosomes. This requirement is satisfied not only by the intact ribosomes but also by stoichiometric amounts of small and large subunits.
5. Two or more enzymatic components (transfer factors and elongation factors) involved in polypeptide chain elongation.
6. Certain cofactors required for chain initiation and termination, as well as for the release of completed chains from their sites of synthesis.
In 1952, P. Siekevitz then working in P.C. Zamecnik’s laboratory at Harvard first discovered a cell-free system for protein synthesis, in which he used the microsomal fraction of rat liver.
Most of the key experiments have subsequently been performed with just two systems:
(i) A cell-free system from rabbit reticulocytes
(ii) a similar system from E. coli and Zamecnik.
All the experimental results with the in vitro systems indicate that protein synthesis consists of four broad sequential steps as follows:
1. Activation of amino acids and formation of amino acyl tRNA molecules
2. Initiation of synthesis of polypeptide chain.
3. Chain elongation.
4. Chain termination.
Let us discuss about the ribosomes and tRNA before describing the process in more detail.
Essay # 2. Ribosomes in Protein Synthesis:
Ribosomes are the organelles of protein synthesis consisting of large and small subunits. A ribosome is a highly specialized and complex structure. From E. coli stable monomeric ribosomes can be obtained in the presence of high concentration of Mg2+, that exhibit a sedimentation coefficient of 70S (Fig. 10.20), a particle having a weight of 25×106, and a diameter of 180 Å.
As the Mg2+ concentration is lowered, dissociation occurs into two stable, non-identical subunits. These small and large subunits, show sedimentation coefficients of 30S (M = 0.85X106 Daltons) and 50S (M = 1.8×106 Daltons), respectively. These subunits can be further split into their constituent proteins (36%) and RNAs (64%).
The 30S subunit contains 21 proteins and a 16S RNA molecule of about 1500 nucleotides of which 11 are specifically methylated. The 50S subunit contains 34 proteins and a large RNA molecule of 23S comprised of about 3000 nucleotides, 22 of which are methylated; as well as a smaller one of 5S species with 120 nucleotides, none of which are methylated.
Protein components of both the subunits are also distinct. The large subunit contains about 34 polypeptides in more or less equimolar amounts. The small subunit contains 21 such polypeptides. All the proteins are different. Only two proteins are common in both the subunits.
Some of these proteins remain attached to the specific regions of RNA providing attachment to other proteins. These proteins are called primary binding proteins. The two subunits can be further dissociated by treatment with 4.9M CsCl containing 0.04M MgCl2 into core particles, lacking some of the proteins, and the proteins in a soluble form called split proteins (SP).
The monomeric unit of eukaryotic ribosome shows a sedimentation coefficient of 77S to 80S and can be dissociated into two asymmetric subunits with sedimentation coefficients of 58S to 60S and ~ 40S (38S to 45S) and molecular weights of 2.4×106 and 1.2×106 respectively.
The subunits contain about 55 per cent RNA and 45 per cent protein. The small subunit contains a single 18S RNA molecule, whereas the large subunit contains three RNA molecules (28S, 7S and 5S). The ribosomal proteins are insoluble in water at pH 7.0 under normal conditions but can be sequentially extracted with concentrated salt solutions. Ribosomes can be reassembled from their component RNAs and proteins.
So far as the active centres of ribosome are concerned, it has got various sites for protein functioning— mRNA binding site, peptidyl (P) site, aminoacyl (A) site, peptidyl transferase site, TU site and EFG site (Fig. 10.21).
mRNA binding site is located on the 30S subunit including the proteins S1, S3, S4, S5, S12, S18, S21 and 3′ end of the 16S rRNA. It is close to the P site. IF3 binds to this site.
P site is mostly located on the 30S subunit and is able to bind initiator tRNA. On the large subunit, the P site includes L2, L14, L18, L24, L27 and L33.
A site is close to the P site. Most of the functions found with the A site are on the 50S subunit. This site includes L1, L5, L7/L12, L20, L30 and L33.
Peptidyl transferase site lies somewhere in the region connecting the A and P sites, close to the terminus of tRNA.
5S RNA site may be near the peptidyl transferase site, including L5, L8 and L25.
TU site includes many proteins like L1, L5, L20 and L7/L12.
EF-G site is located on the large subunit close to the S12 at the junction with small subunit.
Essay # 3. Non-Ribosomal Polypeptide Synthesis:
A different mechanism of protein synthesis is found certain strains of Baccilus brevis, a spore- forming bacterium. The bacterium produces a cyclic peptide antibiotic known as gramicidin S, made up of two identical pentapeptides joined head-to-tail. The biosynthesis of this antibiotic does not depend on the presence of ribosomes or mRNA.
The entire biosynthesis occurs by two enzymes EI and EII. D-phenylalanine is activated by EII, whereas the other four amino acids are activated by EI. The amino acids are activated by the formation of enzyme bound thioesters. The activated amino acid remains attached to a sulfhydryl group of EI or EII.
Similarly, L-proline, L-valine and L-leucine form thioester linkages with specific sulphydryls of Er Likewise, D-phenylalanine forms a thioester bond with EII in the presence of ATP.
The D-phenylalanine residue on EII is transferred to the imino group of the L-proline residue on EI to form a dipeptide.
The activated carbonyl group of the proline residue in the dipeptide reacts with the amino group of the valine residue on the same enzyme to yield a tripeptide. The process is repeated again with ornithine and then with leucine to give a pentapeptide bound to the E1 enzyme. The growing peptide is transferred one by one to different sulphydryl groups with the formation of a peptide bond each time.
Finally, the activated pentapeptides attached to two different EI molecules react with each other to form cyclic gramicidin S.
This mode of protein synthesis is uneconomical compared with the ribosomal mechanism. A peptide containing more than about fifteen residues is not synthesized by this mechanism. Lipmann suggests that this polypeptide synthesis may be a surviving relic of a primitive mechanism of protein synthesis used early in evolution. Ribosomal protein synthesis may have evolved from fatty acid synthesis.
Essay # 4. Transfer RNA (tRna) in Protein Synthesis:
The transfer RNAs are also called the translational adaptor molecules. They occupy an important position in protein synthesis because they provide the link between the nucleotide and the amino acid codes. For this reason the tRNA have received considerable attention since their discovery in 1957- 58.
The base sequence of a tRNA molecule was first determined by Robert Holley in 1965. He also created techniques for the sequencing of nucleic acids in general. Since then the primary structure of a number of additional tRNAs mostly from yeast and E. coli has been elucidated.
The tRNA molecules are relatively small and soluble in 1M NaCl. The length of different species appears to vary from 76 to 85 nucleotide residues.
Their molecular weight is about 25.000. Except for the usual A, G, C and U, they contain a variety of unusual nucleosides, such as pseudouridine (Ψ), dihydrouridine (H2U), ribothymidine (rT), inosine (I), mono and dimethyl derivatives of adenosine and guanosine and thiolated derivatives. These modifications take place on one of the four bases only after it has been incorporated into the polyribonucleotide chain.
From a wide variety of sources the sequences of more than 150 tRNAs have been determined. The single-stranded RNA chain forms a clover leaf structure (Fig. 10.22), due to base pairing between short regions that are complementary.
A standard orientation and terminology for the generalized representation of a tRNA in the clover leaf form are illustrated in the figure. There are four loops and four major arms.
The arms are named for their structure or function. The acceptor arm consisting of a base-paired stem that always ends in an unpaired CCA sequence whose free 2’/3′ OH group is amino-acylated and the 5′ end generally terminates either in G or C.
The other arms consist of paired stems and unpaired loops. The D arm is named for the presence of di-hydrouracil (D) base. The anticodon arm always contains the anticodon triplet in the centre of the loop. The T Ψ C arm is named for the presence of this triplet sequence (Ψ stands for pseudouridine).
The variable feature of tRNA is the so-called extra arm, lying in between the T Ψ C and anticodon arms and on the basis of its nature tRNAs are classified into two types—Class 1 tRNA (having a small extra arm) and the Class 2 tRNA (having a large extra arm).
In clockwise direction around the clover leaf, there are always seven base pairs in the acceptor stem, five in the T Ψ C arm, five in the anticodon arm and usually three in the D arm.
Through X-ray crystallographic studies by Alexander Rich and Aaron Klug the three-dimensional tertiary structure of tRNA is now known to be L-shaped (Fig. 10.23). The base-paired double helical stems of the secondary structure are maintained in the tertiary structure. The arrangement of the stems in three dimensions essentially creates two double helices at right angles to each other.
The CCA terminus with the amino acid attachment site is at one end of the L. The other end of the L is the anticodon loop. The D and TΨC loops form the corner of the L. Most of the bases in the non-helical regions participate in unusual hydrogen bonding interactions which are not usually complementary. This model was worked out in detail for phenylalanine tRNA of yeast, the first tRNA to be crystallized.
This tertiary structure suggests some general conclusions about the function of tRNA. The aminoacyl site is far distant from the anticodon (about 80 Å), which is necessary for the amino acyl group to be located near peptidyl transferase site on the large subunit, while the anticodon pairs with mRNA on the small subunit.
The TΨ C sequence lies at the junction of the arms of the L, possibly for controlling changes in tertiary structure which might occur during protein synthesis. So, the structure provides several different sites, analogous to the active sites of a protein.
Essay # 5. Activation and Formation of Aminoacyl tRNA Molecules in Protein Synthesis:
The twenty amino acids commonly found in protein structure must undergo an initial activation step, which also involves a selection or preliminary screening of amino acids. Peptide bond formation between amino group and carboxyl group of two amino acids is thermodynamically un-favourable.
This barrier is overcome by the activation of carboxyl group of the precursor amino acids.
Each amino acid of the twenty normally found in proteins has its own specific activation enzyme system called aminoacyl tRNA synthetase or ligase or activating enzyme. The activated intermediate is called aminoacyl tRNA which is an ester between the carboxyl group of the amino acid and the 2′ or 3′ hydroxyl group of the ribose unit at the 3′ end of tRNA.
The steps involve the following:
The first step is the formation of an aminoacyl adenylate complex from an amino acid and ATP. This activated complex is a mixed anhydride in which the carboxyl group of the amino acid is linked to the phosphoryl group of AMP.
In the next step, the aminoacyl group is transferred to a tRNA molecule to form aminoacyl tRNA.
The pyrophosphate cleavage of ATP is later on followed by the hydrolysis of the pyrophosphate to yield inorganic phosphate
PPi + H2O → 2Pi
Therefore, two high energy bonds of ATP are used to form a covalent linkage between the amino acid and its corresponding tRNA.
This linkage of an amino acid to a tRNA is important not only because of the activation of carboxyl group to form a peptide, but also because amino acids by themselves cannot recognize the codon on mRNA. Rather, tRNAs bring amino acids to the mRNA-ribosome complex and position them correctly for linking together in the correct sequence.
Essay # 6. Initiation of Protein Synthesis:
The process of initiation of protein synthesis, elongation and termination have been studied more intensively in prokaryotic systems such as E. coli than in other protein-synthesizing entities (Fig. 10.24). The initiation process requires the formation of initiation complex consisting of the 30S ribosomal subunit, mRNA and the three initiation factors found in bacteria, numbered as IF1, IF2 and IF3.
Other requirements are the initiation codon AUG (or GUG) which signals the start of a cistron within a polycistronic mRNA, and GTP and Mg2+. The inadequacy of ribosome subunits in the initiation reaction was first discovered by the effects of washing 30S subunits with ammonium chloride.
The treatment leaves them unable to sponsor initiation of new chains. The reason is that the active 30S subunits contain the initiation factors which are proteinaceous in nature and are bound loosely enough to be released by ammonium chloride wash.
Protein synthesis in bacteria starts with the amino acid formylmethionine (fMet). A special tRNA that brings formylmethionine to the ribosome to initiate protein synthesis, called initiator tRNA (tRNAf) is different from the one that inserts methionine in internal positions (tRNAm).
Methionine is linked to these two kinds of tRNAs by the same aminoacyl tRNA synthetase. A specific enzyme (transformylase) then formylates the amino group of methionine that is attached to tRNAf. The formyl group donor in this reaction is N10-formyl tetrahydrofolic acid.
The first stage of the initiation complex formation involves the binding of the 30S subunit at initiation sites in mRNA with the participation of IF3.
The next stage involves the placement of the initiator fMet – tRNA in the partial P site of the 30S subunit—mRNA complex, with the help of IF2. The role of IF 1 has not yet been known. It may be involved in stabilizing the complex, probably causing a conformational change of the 30S subunit.
After the formation of the initiation complex consisting of the 30S subunit, mRNA, f Met-tRNA and IF 1, IF2 and IF3, the 50S ribosomal subunit becomes associated to form the full 70S initiation complex.
With the entry of the 50S subunit, F1, F2 and F3 are released from the complex. Thus, the initiation factors are concerned solely with the formation of the initiation complex, they are absent from 70S ribosomes and play no part in the stages of elongation.
It has been known for a long time that IF2 has ribosome-dependent GTPase activity, which hydrolyses GTP in the presence of ribosomes, releasing the energy stored in the high-energy bond. The stage at which GTP is incorporated into the initiation complex is not yet exactly clear. Probably it joins after the binary complex (fMet-tRNA and IF2) is associated with the 30S subunit.
With the association of 50S subunit, GTP is cleaved into GDP and P, in an action dependent on the presence of IF2. Now, the P site is occupied by the f-Met-tRNA and the A site is ready to accept the codon- directed incoming aminoacyl tRNA.
In the eukaryotic system, the initiation process is more or less analogous to that in E.coli, but there are at least eight initiation factors found in rabbit reticulocytes, named e IF 1, e IF2, e IF3, e IF4A, e IF4B, e IF4C, e IF4D and e IF5. The prefix ‘e’ indicates their eukaryotic origin. The roles of e IF2 and e IF3 is analogous to IF2 and IF3, respectively. Functions of other factors are not well understood.
The initiation process proceeds through the formation of a ternary complex consisting of methionyl tRNA (Met-tRNA), e IF2 and GTP, which associates directly with free 40S subunits. The association is stimulated by the presence if e IF3 and e IF4C.
The e IF3 facilitates the binding of mRNA with 40S ternary complex. The mRNA binding also requires e IF 1, e 1F4 and e IF4B and ATP. The entry of 60S subunit requires the factor e IF5 and hydrolysis of GTP into GDP and Pi and at this stage all the factors are released from the small subunit.
Chain Elongation:
With the formation of the full 70S initiation complex, the peptide chain elongation can proceed.
The elongation process consists of three steps:
(i) The hydrogen bonding of the appropriate aminoacyl tRNA to the free codon at the A site
(ii) Peptidyl transfer from the peptidyl tRNA (on the P site) to the newly bound aminoacyl tRNA (on the A site)
(iii) Translocation of both the mRNA and the newly synthesized peptidyl tRNA from site A to P and thus the A site is made free for the incoming new aminoacyl tRNA.
Essay # 7. Energetics of Protein Synthesis:
It has been roughly estimated that the standard free energy of the hydrolysis of a peptide bond is about – 5.0 kcal. The process of protein synthesis is an energy-consuming process and, in E. coli, it consumes as much as 90% of the cellular energy.
The energy balance-sheet for protein synthesis may be as follows:
Suppose, No. of amino acid residues in a polypeptide = 200
Then, No. of ATP molecules required to activate amino acids = 200×2 = 400 [2ATPs per molecule of amino acid as to convert the AMP to ADP, one more ATP is required per amino acid]
No. of GTP molecules required during binding of every aminoacyl tRNA to A site = 200
No. of GTP molecules during translocation = 200
... The total number of ~P bonds required = 400 + 200 + 200 = 800
... No. of ~ P bonds required per peptide bond = 2 (from ATP) + 2 (from GTP) = 4
Total amount of energy = 4×7.3 = 29.2 kcal (One ~P – 7.3 kcal) The standard free energy of hydrolysis of one peptide bond = 5.0 kcal
... Balance energy = 29.2 – 5 = 24.2 kcal.
This remaining energy (24.2 kcal) is wasted during the formation of every peptide bond in protein synthesis.