In this blog post, we are going to discuss how protein synthesis in a bacterial cell.
Step by step with a figure.
Before we dive into the process of protein synthesis, you have to understand the basic meaning of protein synthesis. If we talk in terms of molecular biology, protein synthesis is also known as “translation.”
When I read the process of translation, I found something interesting. Why it is called translation? In our day-to-day, life translation means translate any one language into other languages, right?
For example, we just take one sentence in Spanish and translate it into English or any other language. Cell dose the same thing it takes nucleotide language and translates it into protein(polypeptide) language.
I hope you understand the basic idea of protein synthesis. Now let’s see how this process occurs in the cell. Which tools or elements are used in this process?
Approximately 70% of the organic matter of the cell consists of protein. Protein performs a variety of critical functions in the cell as an enzyme, structural protein, or hormones. Protein synthesis is the essential biological process, occurring inside the cell.
It balancing the loss of cellular proteins through the production of new proteins. This protein loss takes place via degradation or exporting outside the cell. Protein is the final product of gene expression.
Let’s take a look at how gene expression is performed in a cell.
The basic concept of the central dogma
If we talk about the central dogma of molecular biology. It explains the flow of genetic information, from DNA to RNA to make a functional product a protein. The concept of central dogma suggests that DNA contains the information needed to make all of our protein, and the RNA is a messenger that carries the information to the ribosome.
The ribosome serves as a factor in the cell where the information is translated from code into a functional product. The simple meaning of gene expression is the process by which DNA instruction is converted into the functional product (protein).
Gene expression has two key stages: 1) Transcription, 2) translation. If you don’t read transcription go and check out our article on transcription. click here.
In this segment, we are going to cover the second stage of gene expression which is translation. Before we going to discuss how protein synthesis occurs let’s take a look at the key component (element) of protein synthesis.
The key element of protein synthesis
There are four main elements that are use in protein synthesis is:
- Activated amino acid
1). Transfer RNA (tRNA) :
To understand how tRNA can serve as an adaptor in translating the language of nucleic acid into the language of protein, we must first examine their structure in more detail. The tRNAs of bacteria have between 73 to 93 nucleotide residues.
Cells have at least one kind of tRNA for each amino acid, at least 32 tRNA is required to recognize all the amino acid codons (Some tRNA recognize more than one codon). But some cells use more than 32.
As shown in the figure, all t RNAs have a hydrogen bonding pattern that forms a cloverleaf structure with four arms. The longer tRNAs have a shorter fifth arm or extra arm. Two of the arms of a tRNA are critical for its adaptor function.
The amino acid arm can carry a specific amino acid esterification by its carboxyl group to the 2′- or 3′- hydroxyl group of the A residue at the 3′ end of the tRNA. The anticodon arm contains the anticodon.
The other major arms are the D arm and TψC arm contribute to important interactions for the overall folding of tRNA molecules, and the TψC arm interacts with the large subunit of rRNA.
2). Activated amino acid
For the translation to occur, a ready supply of tRNA molecules carries the correct amino acid that must be required. Thus, a preparatory step for protein synthesis is amino acid activation. The process in which amino acid is attached to tRNA molecules.
Here, the enzyme called aminoacyl-tRNA synthetases catalyzes amino acid activation. In that case, the amino acid is attached to the high energy bound. The storage of energy in this bond provides the fuel needed to generate the peptide bond when the amino acid is added to the growing peptide chain.
There are at least 20 aminoacyl-tRNA synthetases, each specific for single amino acid and its tRNA. Each tRNA must attach to the corresponding amino acid. Because if an incorrect amino acid is attached to tRNA. It will be incorporated into a polypeptide in place of the correct amino acid.
The protein synthetic machinery recognizes only the anticodon of the aminoacyl-tRNA and cannot tell whether the correct amino acid is attached. Some aminoacyl-tRNA synthetases proofread just like DNA polymerases do.
If the wrong amino acid is attached to tRNA, the enzyme hydrolyzes the amino acid the tRNA, rather than releases the incorrect product.
3). Ribosomes (rRNA)
protein synthesis takes place on ribosomes that serve as workbenches. With mRNA, it is acting as the blueprint. Recall that ribosome is formed from two subunits, the large subunit, and the small subunit.
Here, we discuss the ribosome according to their function in protein synthesis. Bacterial ribosome contains 65% rRNA and 35% protein. The ribosome Can be divided into two functional domains, the first one is the translational domain and the second is the exit domain.
Both subunits contribute to the formation of the translation domain, which interacts with tRNAs and is responsible for forming peptide bonds.
as we see in the figure, The exit domain is located only in the large subunit. Three sites are found within the translation domain for binding tRNA: A, P, and E sites.
The A (aminoacyl or acceptor) site receives tRNA carrying an amino acid required for the protein synthesis. The P (peptidyl or donor) site holds a tRNA attached to the growing polypeptide. The E (exit) site is the location from which empty tRNAs leave the ribosome.
Ribosomal RNA (rRNA) is thought to have three roles.
- All their rRNA molecules contribute to the ribosome structure
- The 16S rRNA of the 30S subunit is needed for the initiation of protein synthesis because its 3′ end binds to a site on the leader of the mRNA called the Shine-Dalgarno sequence. Thus, the Shine-Dalgarno sequence of part of the ribosome-binding site (RBS). This helps rRNA to position on the ribosome. The 16S rRNA also bind the protein needed to initiate translation (initiation factor 3)
- The 23S rRNA is a ribosome that catalyzes peptide bond formation.
4). Messenger RNA (mRNA)
Messenger RNA (mRNA) is the molecule in the cell that carries codes from the DNA to the sites of protein synthesis in the ribosomes. Because the information in DNA cannot be decoded directly into protein. It is first transcribed into mRNA (see Transcription).
Each molecule of mRNA encodes the information for one or more proteins in bacteria. For more detail see transcription
Steps of protein synthesis
1). Initiation of protein synthesis.
The initiation of protein synthesis is very complex. The complexity is necessary to ensure that the ribosome does not start synthesizing a polypeptide chain in the middle of a gene. The initiation of protein synthesis occurs in three steps.
First step: formation of 30S complex
In the first step, the 30S ribosomal subunit binds two initiation factors (IF), is IF1 and IF3. The factor IF3 prevents the 30s and 50s ribosomal subunits from combining prematurely.
The mRNA then binds to the 30s subunit. This is accomplished with the help of the 16S rRNA within the 30S subunit. Which is complementary to the AUG codon and binds the Shine-Dalgarno sequence in the leader sequence of the mRNA.
This consensus sequence is an initiation signal of 4 to 9 purine residues. The sequence base pairs with a complementary pyrimidine-rich sequence near the 3′ end of the 16S rRNA of the 30S ribosomal subunit.
This mRNA – rRNA interaction positions the initiating (5′) AUG sequence of the mRNA in the precise position on the 30S subunit where it is required for initiation of translation. The reason, I’m writing (5′) AUG is because the RNA sequence reads the 5′ to 3′ direction.
Bacteria begin protein synthesis with a modified aminoacyl-tRNA, N- formyl methionyl-tRNAfMet (fMet-tRNA). Which is called the initiator tRNA and is coded by the start codon AUG.
The amino acid of the initiator tRNA has a formyl group covalently bound to the amino group and can be used only for initiation because of the presence of the formyl group. When methionine is to be added to a growing polypeptide chain, a normal methionyl-tRNAMet is employed.
Although the formyl group is not retained but is hydrolytically removed. One to three amino acids may be removed from the amino terminus of the polypeptide after synthesis.
As we discussed above bacterial ribosomes have three sites that bind with tRNA, the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site.
The A and P sites bind amino aminoacyl-tRNA, whereas the E site binds only uncharged tRNA that has completed their task on the ribosome. Both 30S and 50S subunits contribute to the characteristics of the A and P sites, whereas the E site is largely confined to the 50S subunit.
The initiating codon AUG is positioned at the P site, the only site to which fMet-tRNAfMet can bind. The fMet-tRNA met is the only aminoacyl-tRNA that binds first to the P site during the subsequent elongation stage, all other incoming aminoacyl-tRNA binds first to the A site and only subsequently to the P and E sites.
The E site is the site from which the uncharged tRNA sleaves during elongation. Factor IF1 binds at the A site and prevents tRNA at this site during initiation.
Second step : Pairing with initiation codon.
In the second step of the initiation process, the complex consisting of the 30S ribosomal subunit, IF3, and mRNA is joined by both GTP- bound IF2 and the initiating fMet-tRNAfMet. The anticodon of this tRNA now pairs correctly with the mRNA’s initiation codon.
Third step : Formation of large 70S complex
In the third step, this large complex combines with the 50S ribosomal subunit. Simultaneously, the GTP-bound IF is hydrolyzed to GDP and Pi(pyrophosphate), which are released from the complex. All three initiation factors leave the ribosome at this point.
Completion of these steps produces a functional 70S ribosome called the initiation complex, containing the mRNA and the initiating fMet-tRNAfMet. The correct binding of the fMet-tRNAfMet to the P site in the complete 70S initiation complex is ensured by at least three points of recognition and attachment
- AUG fixed in the P site.
- The interaction between the Shine-Dalgarno sequence in the m RNA and the 16S rRNA.
- The interaction between the ribosomal P site and the fMet-tRNAfMet.
The initiation complex is now ready for elongation.
2). Elongation of protein synthesis.
The second stage of protein synthesis is elongation. Cells use three steps to add amino acid residue, and the step is respected as many times as there are residues to be added.
Elongation step 1: Binding of an incoming Aminoacyl-tRNA
In the first step of the elongation cycle, the appropriate aminoacyl-tRNA binds to a complex of GTP- bound EF-Tu (Elongation Factor Thermo Unstable). EF-Tu is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA to the ribosome.
The resulting aminoacyl-tRNA-EF-Tu-GTP complex binds to the A site of the 70S ribosome. The EF-Tu-GTP complex is regenerated in a process requiring EF-Ts and GTP.
Elongation step 2: Peptide Bond Formation
A peptide bond is now formed between the two amino acids bound by their tRNAs to the A and P sites on the ribosome. This occurs by the transfer for the initiating N-formyl methionyl group from its tRNA to the amino group of the second amino acid, now in the A site.
The α-amino group of the amino acid in the A site acts as a nucleophilic, displacing the tRNA in the P site to form a peptide bond. This reaction Produces a dipeptidyl- tRNA in the A site, and now “uncharged” (deacetylated) tRNA remains bound to the p site.
The tRNA then sift to a hybrid binding state, with the element of each spanning two different sites on the ribosome The enzyme activity that catalyzes peptide bond formation has historically been referred to as peptidyl transferase and was widely assumed to be intrinsic to one or more of the proteins in the large ribosomal subunit.
We now know that this reaction is catalyzed by the 23s rRNA, adding to the known catalytic repertoire of ribosomes.
Elongation step 3: Translocation
In the final step of the elongation cycle is translocation, the ribosome moves one codon toward the 3′ end of the mRNA. This movement shifts the anticodon of the dipeptidyl-tRNA, which is still attached to the second codon of the mRNA, from the A site to the P site, and shifts the uncharged tRNA from the P site to the E site.
From where the tRNA is released into the cytosol. The third codon of the mRNA now lies in the A site and the second codon in the P site. Movement of the ribosome along the mRNA requires EF-G (also known as translocase) and the energy provided by the hydrolysis of another molecule of GTP.
A change in the three-dimensional conformation of the entire ribosome results in its movement along the mRNA. Because the structure of EF-G mimics the structure of the EF-Tu-tRNA complex, EF-G can bind the A site and presumably, displace the peptidyl-tRNA.
After translocation, the ribosome, with its attached dipeptidyl-tRNA and mRNA, is ready for the next elongation cycle and attachment of a third amino acid residue.
For each amino acid residue correctly added to the growing polypeptide, two GTPs are hydrolyzed to GDP and Pi as the ribosome moves from codon to codon along the mRNA toward the 3′ end.
The polypeptide remains attached to the tRNA of the most recent amino acid to be inserted. This association maintains the functions connection between the information in the mRNA and its decoded polypeptide output.
At the same time, the ester linkage between this tRNA and the carboxyl terminus of the growth of the growing polypeptide activates the terminal carboxyl group for nucleophilic attack by the incoming amino acid to form a new peptide bond.
As the existing ester linkage between the polypeptide and tRNA is broken during peptide formation. The linkage between the polypeptide and the information in the mRNA persists because each newly added amino acid is still attached to its tRNA.
3).Termination of protein synthesis
Elongation continues until the ribosome adds the last amino acid coded by the mRNA.
Termination of the last stage of polypeptide synthesis is signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA), immediately following coded amino acid. Mutation in a tRNA anticodon that allows an amino acid to be inserted at a termination codon is generally deleterious to the cell.
In bacteria, once a termination codon occupies the ribosomal A site, three termination factors or release factors (RF) the protein RF1, RF2, and RF3 contribute to.
- Hydrolysis of the terminal peptidyl-tRNA bound.
- Release of the free polypeptide and the last tRNA, now uncharged, from the P site
- Dissociation of the 70S ribosome into its 30S and 50S subunits, ready to start a new cycle of polypeptide synthesis.
RF1 recognizes the termination codons UAG and UAA, and RF2 recognizes UGA and UAA. Either RF1 or RF2 binds at termination codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid.
The specific function of RF3 has not been firmly established, although it is thought to release the ribosomal subunit. Ribosome recycling leads to the dissociation of the translation components. The release factors dissociate from the post-termination complex and are replaced by EF-G and a protein called ribosome recycling factor.
Hydrolysis of GTP by EF-G leads to dissociation of the 50S subunit from the 30S-tRNA-mRNA complex. EF-G and ribosome release factors are replaced by IF3, which promotes dissociation of the tRNA.
The mRNA is then released. The complex of IF3 and the 30S subunit is then ready to initiate another round of protein synthesis.