In this article we will discuss about the Mechanism of Protein Synthesis in Prokaryotes and Eukaryotes.
Protein synthesis in the cell is conducted by ribosomes that are found attached to the membrane of endoplasmic reticulum and microsomes, as well as in free state in the groundplasm.
The main components that take part in protein synthesis at cellular level are: 20 different amino acids, different types of RNAs, enzymes, aminoacid activating enzymes, polypeotide-polymerase and energy liberating molecules, such as ATP and GTP.
DNA which contains genetic information synthesizes three kinds of RNA:
(i) Messenger RNA (mRNA)
(ii) Ribosomal RNA (rRNA) and
(iii) Transfer RNA (tRNA) or soluble RNA (sRNA).
mRNA is copied from DNA molecule. The specific locus of DNA molecule where mRNA is formed is referred to as a structural gene. tRNAs come probably from special genes called determinants for tRNAs.
A Protein Synthesis in Prokaryotes:
The mechanism of protein synthesis has been thoroughly investigated in Escherichia coli. In bacterial cell, the protein synthesis takes place on 70s ribosomes.
The process of protein synthesis in E. coli involves the following steps:
The partial uncoiling of two DNA strands occurs. This is followed by the production of single stranded mRNA on one of the two DNA strands. The messenger RNA complement is made in accordance with base pairing rules. This is transcription.
The transcription of genetic code of DNA into mRNA is catalysed by the enzyme RNA polymerase. mRNA carries the information in the form of base triplets for the synthesis of a particular protein (Fig. 20.2).
2. The second step involves the separation of mRNA from DNA and then its transfer from nucleus to cytoplasm and final attachment of 5′ end of mRNA with 30s (smaller) sub-unit of ribosome in presence of protein initiation factor. Before the mRNA migrates from nucleus to ribosome in cytoplasm it undergoes process of maturation.
In eukaryotes the newly formed RNA is called heterogenous nuclear RNA (hn RNA). Many of the functional RNA molecules including ?RNA and mRNA in eukaryotes are processed from much longer precursor RNAs which are 5,000 to 50,000 nucleotides long and may be 10 to 100 times longer than the mature functional RNA molecules which are derived from them.
Precursor RNAs are transcripts of split genes which contain both sequences coding for aminoacids (exons) and those not coding for aminoacids (introns) interspersed. The non-coding sequences from the pre-RNAs are cleaved out and coding sequences are spliced together to produce functional mature RNA molecules.
Few of the eukaryotic genes are not split. In prokaryotes some of the RNA molecules are cleavage products of longer pre-RNA.
As has been pointed out, mRNA determines the sequence of amino acids in the polypeptide chain which in turn is determined by sequence of nucleotides in DNA (gene). This process is called translation. Translation involves the following steps which are shown in Figs. 20.2, 20.3, 20.4.
(a) Activation of aminoacid:
Elsewhere in the cytoplasm, aminoacids are selected for activation. Professor Fritz Libmann and others discovered in 1956 that before amino acids can combine to form proteins, they must be activated and this is achieved by combining with phosphate. The activation involves the reaction between aminoacid and ATP.
The reaction is catalysed by specific enzyme aminoacyl RNA synthesize. So, for the activation of 20 aminoacids there should be at least a set of 20 such enzymes in the cytoplasm. Amino acid activating enzymes were first discovered by M. Hoagland.
The product formed after activation is aminoacyl-adenylate enzyme complex which is energy rich compound.
(b) Attachment of activated aminoacid to tRNA:
The CCA end of tRNA molecule now attaches with specific aminoacid adenylate-enzyme complex. The aminoacyl-adenylate remains attached to the enzyme in the form of monocovalent complex until it is transferred to tRNA. The carboxyl group of aminoacid residue of aminoacyl adenylate is transferred to 3′ OH group of ribose sugar of terminal adenosine at CCA end of tRNA. As a result, aminoacyl-tRNA, AMP and enzyme are formed.
The transfer of aminoacids to tRNA is catalysed by the previous aminoacyl RNA synthetase enzyme itself (Fig. 20.3).
(c) The aminoacids – tRNA complex then comes to mRNA where adapter nucleotide triplet or anticodon of tRNA becomes attached with the complementary base triplet (codon) of mRNA.
The fate of amino acid is determined at the very moment, it becomes attached with the corresponding tRNA. The messenger RNA and tRNA-amino acid complex attachment is temporary. Like this, many /RN A-amino acid complexes are arranged one after another at proper places on messenger RNA strand in linear fashion. In the attachment, the adapter trinuceleotides of rRNAs act as anticodons (Figs. 20.4 and 20.5).
The mRNA molecules have translation initiation site at 5′ end and the chain termination site close to trailor end. The initiation site consists of a codon AUG and unknown secondary structure of mRNA. The chain termination site has one of the three codons UAA, UAG and UGA.
Mature mRNA binds with smaller ribosomal sub-unit in presence of initiation factor IF2. Soon tRNA-N-Formyl methionine complex (F met-tRNA) comes from the cytoplasmic amino acid pool and binds with the first triplet codon of mRNA to initiate the process of protein synthesis and to form initiation complex.
Initiation complex is formed in presence of guanosine triphosphate (GTP) and three protein factors F1, F2 and F3. This is followed by union of bigger sub-unit with smaller ribosomal sub-unit in presence of Mg++ and initiation factors F1, F2 to form the ribosome. The codes of mRNA are not read by a single ribosome but by many ribosomes interlinked by mRNA (Polysomes).
(d) Initiation of Protein Synthesis:
The messenger RNA always has first triplet as AUG or GUG at its 5-end and these triplets code for aminoacids N-formyl methionine (F. met) which usually initiates a protein chain.
Thus, in all proteins formyl methionine occupies the first place, i.e., at, amino end and when the protein molecules are completely synthesised formyl methionine may be detached from the protein molecules by activity of hydrolytic enzyme deformylase.
In formyl methionine- tRNA complex the amino group is blocked by formyl group leaving only COOH group free to react with NH2 group of the second amino acid (AA2). In this way, polypeptide chain always grows from amino end toward-COOH end.
When one tRNA-aminoacid complex attaches to mRNA at starting end, then the second tRNA-aminoacid complex also comes just after the first and finally the two adjacent amino acids form peptide linkage. Like this several molecules of amino acids will join in a definite order through peptide bonds to form specific protein molecule (Fig. 20.5).
(e) Elongation of Polypeptide Chain:
The peptide chain elongates by regular addition of aminoacids and relative movement of ribosome along with messenger RNA in presence of GTP (guanosine triphosphate) in the following sequence:
(a) According to W.D. Stansfield (1969) there are three presumed sites in the ribosome Figs. 20.3 and 20.4. These are:
(i) Decoding site or ‘A’ site which binds the loaded AA~tRNA complex with the mRNA by base pairing.
(ii) A condensing site or ‘P’ site or peptidyl site which joins the aminoacid to the growing polypeptide chain.
(iii) An exit site or ‘E’ site at which tRNA detaches from the polypeptide, messenger RNA and ribosome.
tRNA with their associated aminoacids will enter the ribosomal site ‘A’ and will be checked by a ‘checking factor’ to see if there is a correct fit between the codon on the messengers RNA and the anticodon of tRNA. If the fit is incorrect, the tRNA is rejected and presumably other /RNAs will continue to be tried until the correct one is found.
(b) As the initiating codon AUG or GUG has entered the ribosome and is in position facing the site ‘A’, the correct tRNA, i.e., f-met-tRNA is checked against codon. This reaction is facilitated by the presence of initiation factor F1
(c) The 30S f-met-tRNA along with messenger RNA, then moves from decoding site (‘A’ site) to peptidyl site (‘P’ site) and with this the next codon of mRNA enters ‘A’ site where it finds the second correct aminoacyl-tRNA. Aminoacyl-tRNA (AA2 – tRNA) binds with the codon of ‘A’ site in presence of GTP and two proteins called transfer factor Tu and Ts which remain associated with ribosomes.
In this binding process, a complex is formed from GTP, the transfer factors and the incoming aminoacyl-tRNA which ultimately fixes aminoacyl-tRNA (AA2 tRNA) at the ‘A’ site of ribosome and at the same time releases transfer factors – GTP complex and inorganic phosphate.
(d) Due to the relative movement of ribosome and mRNA in presence of single GTP molecule the next codon enters the ‘A’ site. The A site is now occupied by another aminoacyl- tRNA (AA3– tRNA) corresponding to the next codon of mRNA and f-met-tRNA reaches at the exit site (E-site) and AA2-tRNA occurs at the P site. Now an enzyme known as transferase I kicks off tRNA from formyl methionine and flips the formyl methione (AA1,) to AA2-tRNA bound at the ‘P’ site.
According to Monro (1967) an enzyme known as peptidyl synthetase found in SOS, sub-unit helps in the formation of peptide bond. The ‘G’ factor is supposed to release the discharged or deacetylated tRNA from the site ‘E’ of ribosome.
(e) The next stage of elongation process follows that involves establishment of peptide bond by reaction between free NH2 group of incoming amino acid and carboxyl group of the polypeptide.
Thus, during the elongation of polypeptide chain, each charged tRNA (aminoacyl-tRNA) enters the decoding site, moves to ‘P’ site, transfers its aminoacid to the carboxyl end of polypeptide, moves to exit site where polypeptide chain is transferred to adjacent tRNA bound at ‘P’ site and then finally released from the ribosome.
The synthesis of polypeptide chain is completed according to the codons of messenger RNA and the process comes to an end abruptly where any one of the three non-sense triplets UAG, UAA and UGA is present in the messenger RNA. Generally, no tRNA has anticodon for any of these three ‘nonsense codons’ but some suppressor mutations produce tRNA with any of these three codons.
Dissociation of Initiation Factors from the Initiation Complex:
The polypeptide chain, still bound to the /RNA is attached to mRNA. The chain is released from the ribosome under the direction of three distinct proteins which are called released factors R1, R2 and S.
These factors are bound to the ribosome and control the hydrolysis of ester linkage between tRNA and the polypeptide chain. Reproduction of a primary polypeptide chain according to specification of mRNA is called translation.
After the completion of chain the two sub-units of ribosomes separate.
Protein Synthesis on 80S Ribosomes of Eukaryotes:
The process of protein synthesis on SOS ribosomes of eukaryotes is found to be more or less similar to that on 70S ribosomes described above.
However, the process of initiation of polypeptide chain on 8OS ribosomes of eukaryotes differs from that of prokaryotes in the following two aspects:
1. In eukaryotes in place of formyl methionine methionine acts as chain initiation aminoacid.
2. In eukaryotes smaller sub-unit (40S) associates with methionine-tRNA without the help of mRNA.
3. For the formation of initiation complex involvement of GTP is not necessary (Zasloff and Ochoa, 1972).
4. The initiation process involves the following steps:
(i) Met-/RNA + 40S sub-unit———- > 40S-met rRNA
(ii) 40S-met /RNA + mRNA———- > 40S-mRNA-met /RNA
(iii) 40S-mRNA-met /RNA + 60S sub-unit———– > 80S-mRNA met-tRNA initiation-complex.