As an extremely broad scientific field, Biochemistry comprises a wide range of interesting and exciting concepts that explain the majority of the processes occurring in a living organism. One of such concepts is referred to as protein synthesis – the way proteins are produced in prokaryotic or eukaryotic cells. The process is relatively straightforward and rapid in the case of prokaryotic cells lacking membrane-bound organelles. On the other hand, protein synthesis is a much more complicated process in the case of eukaryotes.
The actual process of protein synthesis takes place in the rough endoplasmic reticulum (RER) employing ribosomes that are attached to the reticulum. That is why the primary function of RER is to produce proteins. Although, the whole process is much more complicated than it seems at first glance.
Protein synthesis is generally divided into two parts: transcription and translation. It is crucial to examine both processes in order to get an idea of how the whole process really works. Therefore, to make it easier for you to explore the concept, the article will provide major aspects of protein synthesis in prokaryotes as well as eukaryotes.
Transcription in Prokaryotes
Since prokaryotes lack membrane-bound organelles, transcription in such organisms occurs in the cytoplasm of the cell. RNA polymerase (abbreviated as RNAP) is the enzyme that is responsible for the synthesis of RNA. Prokaryotes utilize the same RNAP to transcribe the genes. The reaction proceeds in the way that the RNAP couples NTPs (ribonucleoside triphosphates: ATP, CTP, GTP, UTP) on DNA templates resulting in the release and hydrolysis of PPi (free phosphate groups). The reason for the RNAP involvement in the process is that it does not require a primer, while DNA polymerases do.
The transcription process can be further divided into three major processes:
Transcription starts with the recognition of a proper DNA strand to initiate RNA synthesis. Meaning that RNAP binds to the site of the initiation via protomers, which are base sequences. The polypeptide units assemble and disassemble once a particular gene is transcribed, and the process is complete. Stable complexes are formed between the holoenzyme (polymerase with all 5 subunits) and protomer molecules. The rate of gene transcription is dependent upon the speed at which the protomers form stable complexes with RNAP holoenzyme.
One of the DNA strands is transcribed, representing a template strand from which mRNA is produced. mRNA strand is almost identical as the non-template DNA strand with one exception: the mRNA strand consists of uracil (U) instead of thymine (T) that is present in DNA. RNAP then adds new nucleotides meaning that mRNA grows from the 5’ to 3’ end.
Hydrogen bonds are broken and reformed through the process of elongation. As a result, DNA is unwound and rewound continuously. RNAP plays a vital role in the process since it guarantees that elongation is not intervened before the designated time.
As suggested by electron micrographs, there are specific sites of DNA at which transcription is terminated. Although in some cases there is no requirement for the involvement of a protein known as Rho Factor (the situation is referred to as a Rho-independent termination), termination often requires the action of this factor to finish the transcription process. Being a hexamer of 419-residue subunits (identical), the Rho factor improves the termination process and supports spontaneous termination of transcription. The way the factor works is the following: Rho factor recognizes a particular sequence in RNA and attaches to it while migrating in the 5’-3’ direction until it gets to the point where RNAP has paused at the termination site.
At this point, the transcription process is complete. Transcription and translation co-occur in prokaryotic cells, meaning that the translation process begins and proceeds even if the synthesis of mRNA has not been finished yet.
Transcription in Eukaryotes
Principle aspects of the transcription process in prokaryotes and eukaryotes are somewhat similar. But there are still some significant differences that make the two processes unique. One of the major disparities is that eukaryotic cells comprise several RNAPs, each responsible for the synthesis of a different group of RNA. Therefore, the sequences are controlled in a much more sophisticated manner. Along with that, eukaryotic cells require more than 100 polypeptides to initiate transcription.
Similarly to prokaryotic transcription, transcription in eukaryotes also proceeds in three major steps which are the following:
Initiation in eukaryotic cells requires RNAPs similarly to prokaryotic transcription. The major difference between prokaryotes and eukaryotes is that eukaryotes comprise several types of RNAPs – RNAP I, RNAP II, and RNAP III. Each RNAP has a distinct location and is responsible for the specific RNA synthesis:
- RNAP I – located in the nucleoli, RNAP I is responsible for the synthesis of the rRNA precursors.
- RNAP II – located in the nucleoplasm, RNAP II is responsible for the synthesis of the mRNA precursors.
- RNAP III – located in the nucleoplasm, RNAP III synthesizes the 5S rRNA precursors, tRNAs, and some other cytosolic and nuclear RNAs.
Along with these RNAPs, eukaryotes consist of other mitochondrial or chloroplast (plants) RNAPs.
If RNAP in prokaryotes could bind directly to a DNA template without the involvement of other molecules, eukaryotic RNAPs require the assistance of different protein molecules that are called transcription factors. These transcription factors bind to the specific region of a protomer and then support the proper recruitment of RNAP.
The core segment of a protomer that represents the binding site for a transcription factor is referred to as a TATA box. But if promoters lack the TATA box (nearly all promoters recognized either by RNAP I or RNAP III), they bind to TBP (TATA-binding protein), which is a universal transcription factor.
After RNA synthesis has been successfully initiated and a short transcript has been produced, the transcription proceeds to the elongation mode. Eukaryotic elongation requires the involvement of a wide range of factors. The elongation process involves displacement of the “finger domain” of TFIIB and phosphorylation of the C-terminal domain of RNAP II’s subunit (Rpb1). As a result, phosphorylated RNAP II releases transcription-initiating factors.
Finally, the elongator complex (six-protein complex that accelerates the transcription process) binds to the phosphorylated C-terminal domain of Rpb1 in the place of ejected transcription factors.
Synthesis of new RNA strands is catalyzed by RNAPs in the 5’-3’ direction, meaning that new nucleotides are added to the 3’ end of the RNA strand. The sequence of the DNA strand governs the nucleotide addition. The process of elongation is transferred to the next DNA nucleotide on the template strand until the transcription is terminated.
In the case of eukaryotic cells, there are no precise termination sites. Although, termination of the transcription process occurs when RNAP transcribes a specific sequence of DNA strand, which represents a stop signal known as a terminator. Since the primary transcripts of a particular DNA strand have heterogeneous sequences, it has not been possible to identify termination sequences in eukaryotes accurately.
Even though transcription termination is an imprecise process, there is no need for specific termination sites. The reason for this is the endonucleolytic cleavage at a particular location that is involved in transcript processing, which means that RNA cleavage might act as a signal for the polymerase to terminate transcription.
Translation and Protein Synthesis in Eukaryotes
After the sequence of DNA is transcribed into the sequence of RNA, the translation process occurs. Translation of mRNA is the process that results in the identification of the amino acid sequence of a protein being synthesized.
Ribosomes are responsible for the orchestration of the translation of mRNA to the synthesis of polypeptides. There are several significant concepts that you should know before proceeding to further examination of the process:
- Polypeptides are synthesized int the following direction: N-terminus à C-terminus
- Chain elongation proceeds by linking the polypeptide to the translated tRNA’s amino acid residue.
- Ribosomes translate mRNA sequence in the 5’-3’ direction.
- Active translation takes place on polyribosomes (polysomes).
Similarly to transcription, translation process also proceeds in three primary steps:
The first step in protein synthesis is the chain initiation that requires the involvement of tRNA and initiation factors. tRNA that is involved in the initiation process recognizes the initiation codon (triplet that is necessary for a single amino acid specification) and is referred to as a tRNAfMet. Moreover, ribosomal translation initiation in eukaryotes requires a minimum of 12 soluble protein, each consisting of 26 polypeptide chains.
The next step in the protein synthesis is referred to as the polypeptide chain elongation, which proceeds in three stages and requires non-ribosomal elongation factors:
In the stage of decoding, the ribosome selects and binds a specific aminoacyl-tRNA if its anticodon is complementary to the mRNA codon located in the A site.
The transpeptidation process is also referred to as a peptide bond formation, which occurs as a result of peptidyl group transfer from the P-site tRNA to the aminoacyl group in the A Site.
During this stage, A- and P-site tRNAs are transferred to P and E sites accordingly along with their bound mRNA.
Translation of synthetic and natural mRNAs differ significantly. In the case of synthetic mRNA translation, there is a consequent peptidyl-tRNA “stuck” in the ribosome. On the other hand, translation of natural mRNA results in the production of free polypeptides due to the fact that natural mRNAs comprise stop codons (e.g., UAA, UGA, UAG).
Therefore, the involvement of release factors (RFs) is essential to terminate the translation process. Even though the RFs are quite similar to each other, they still recognize different stop codons. For instance, RF1 and RF2 (in prokaryotes) are 39% identical, but RF1 recognizes UAA and UAG, while RF2 recognizes UAA and UGA stop codons. On the contrary, there is a single release factor known as eRF1 in eukaryotes that recognizes all three stop codons (UAA, UGA, and UAG).
As the termination of the translation process is complete, proteins are synthesized.
To sum up, protein synthesis involves 2 major steps: transcription and translation. Each step comprises an additional 3 stages, which are initiation, elongation, and termination. Transcription occurs in the nucleus and results in the production of mRNA molecules, while translation proceeds in the cytoplasm on the rough endoplasmic reticulum through the involvement of ribosomes.