The genetic information is present in all the organisms in the form of DNA. This DNA is present in the nuclei of eukaryotic cells and contains the information for all the traits expressed by the cell. The genetic material controls al the traits of a cell by guiding and controlling the synthesis of proteins.
Gene expression is the process by which proteins are synthesized from the information present in the DNA. This process consists of two phases;
- Transcription of DNA to form RNA
- Translation of mRNA to form proteins
The genetic expression or gene expression is also sometimes called the central dogma of the cell. in this article, we will discuss different steps of gene expression as well as its regulation.
The region of DNA that codes for one particular protein is called a gene. The nucleotide sequence present in a gene is copied into the nucleotide sequence of mRNA. This mRNA molecule serves as a transcript of that particular gene. This process of making an RNA transcript of a gene is called transcription.
It should be kept in mind that all types of RNA are made from DNA via the process of transcription.
The process of transcription requires certain enzymes called RNA polymerases. These enzymes read the nucleotide sequence of the gene and construct RNA molecules with a complementary nucleotide sequence using new nucleotides.
The p[prokaryotic cells have only one type of RNA polymerase that synthesizes mall types of RNA. However, eukaryotic cells have three types of RNA polymerase;
- RNA polymerase I for rRNA
- RNA polymerase II for mRNA
- RNA polymerase III for tRNA
All types of RNA are first generated in the form of a linear poly-ribonucleotide chain. These chains later undergo post-transcriptional modification to get their final shape.
In this article, we will only discuss mRNA in detail.
Coding strand and Template strand
Recall that DNA is a double-stranded molecule while RNA is only single-stranded. During the process of transcription, the nucleotide sequence of only one strand can be used to make RNA.
The DNA strand whose nucleotide sequence is used to make a complementary nucleotide sequence of RNA is called the template strand or non-coding strand. the nucleotide sequence of this strand is used as a template by RNA polymerase to make RNA.
The opposite strand, that is not read by RNA polymerase, is called a non-template strand or coding strand. It is called so because the nucleotide sequence of this strand directly corresponds to the nucleotide sequence of RNA i.e., they both have identical nucleotide sequences except the difference of thymine and uracil.
It should be kept in mind that the same strand can serve as a template strand for one gene and coding strand for some other genes.
Steps in Transcription
The process of transcription involves the following steps.
It the first step in the transcription of a gene. During this process, the RNA polymerase enzyme recognizes and binds to a particular nucleotide sequence in the promoter region of the gene. The promotor region is present upstream of the gene and is not transcribed. The special sequence that is recognized by RNA polymerase is called the Consensus sequence. It is different in prokaryotes and eukaryotes.
The consensus sequence in prokaryotes are;
Eukaryotes can have the following consensus sequence;
- -TATA- sequence
- -GC- sequence
- -CAAT- sequence
After the binding of RNA polymerase to the promoter region, the DNA double helix is unwound. Once the polymerase reaches the transcription initiation site, it starts constructing the ribonucleotide chain using ribonucleotide triphosphates. The sequence of this chain is complementary to the template strand that is being read by RNA polymerase. The transcription always occurs in 5’ to 3 ‘ direction. The new strand is constructed in 5’ to 3’ direction while the template strand is read in 3’ to 5’ direction.
The unwinding of DNA during this process generates supercoils that are later removed by DNA topoisomerases.
The elongation of the ribonucleotide sequence continues until a special sequence is reached called the termination sequence. The RNA polymerase halts once it reaches the termination sequence. Two types of termination are usually seen, rho-independent or rho-dependent termination.
- Rho-independent termination: In this case, a sequence at the end of the gene creates a self-complementary sequence in the newly constructed RNA. It causes the DNA to fold back onto itself forming a structure resembling a hairpin, called hairpin loop. This hairpin causes the RNA to separate from the DNA.
- Rho-dependent termination: This type of termination is facilitated by a protein called rho-protein. This protein has an intrinsic ATPase activity. It binds to the 5’ end of the new RNA chain and travels down till it reaches the 3’ end. Here, it uses ATP to separate the DNA and RNA strands.
Transcription factors, Transcription activators, and Enhancers
In the case of eukaryotes, the RNA polymerase does not itself recognize and bind the promoter sequence. Rather, it requires some special proteins that assist in recognizing and binding to the promoter region. These are called general transcription factors.
They are made in the cytosol and are carried to the nucleus for their action.
Transcription activators are the special transcription factors that recognize and bind to special sequences located upstream of the promoter region. They play an important role in regulating the process of transcription.
Enhancers are the DNA sequences that can increase the rate of transcription if bound to special transcription factors. They also play a role in the regulation of gene expression.
The linear ribonucleotide chain made as a result of the above process undergoes modifications to make functional copies of RNA.
- Ribosomal RNA
The rRNA is made from long precursor molecules. These molecules are cleaved to smaller segments that are then joined to form rRNA.
- Transfer RNA
The ribonucleotide chain undergoes following modifications to form tRNA;
- Removal of 16 nucleotide sequence from 5’ end
- Removal of 14 nucleotide sequence from anticodon loop
- Modification of some bases
- Replacement of uracil residues at the 3’ end by CCA sequence
- Messenger RNA
The prokaryotes do not have any nucleus. Their DNA is made in the cytoplasm and thus needs no post-translational modifications. On the other hand, eukaryotic mRNA is made in the nucleus. It must undergo some modification so that it can be sent to the cytoplasm for proper functioning. These modifications include the following;
- 5’ Cap: A molecule of GTP is added via 5’-5’ triphosphate linkage at the 5’ end. This molecule serves as a cap, protecting the 5’ end from the action of exonucleases.
- Poly-A tail: A sequence of around 20 adenine nucleotide is added to the 3’ end. It serves as a tail, protecting the 3’ end from the action of exonucleases.
The mRNA thus formed travels to the cytoplasm where it can participate in the next step of gene expression i.e. translation.
This is the process by which the information in the nucleotide sequence of mRNA is translated into the sequence of amino acids in a protein. The process of translation takes place as a result of the interaction between all the three major types of RNA. The information for protein synthesis is present in the mRNA in the form of a genetic code that is read by the anticodon loop of tRNA.
Genetic Code and Anticodon
Genetic code is a sequence of three nucleotides that codes for one specific amino acid in protein. One genetic code is also called a codon. A codon is regarded as one word made up of three alphabets. The three alphabets are three nucleotides that are read together.
The codon is read and decoded into the sequence of amino acids by tRNA. The anticodon loop of tRNA also has a sequence of three nucleotides. This sequence is complementary to the codons and is thus called the anticodon sequence.
Role of tRNA
As mentioned earlier, the anticodon loop of tRNA reads the genetic code and inserts the specific amino acid in the polypeptide chain. One tRNA can only carry one specific amino acid. The amino acid is attached to the hydroxyl group of the nucleotide at the 3’end of tRNA. A molecule of tRNA having the specific amino acid attached to its 3’end is called charged tRNA.
The charging of tRNA is done by a specific enzyme called aminoacyl tRNA synthetase. This enzyme uses ATP and amino acid to form an enzyme-AMP-aminoacid complex. In the next step, the amino acid is transferred from this complex to the molecule of tRNA.
The enzyme is extremely specific for tRNA and amino acid. There is one specific enzyme for each type of amino acid, just like tRNA.
Role of Ribosomes
Ribosomes are the machinery for protein synthesis. These are the small organelles present in the cytoplasm or attached to the rough endoplasmic reticulum (only in eukaryotes). Ribosomes are made up of ribosomal RNA and proteins. They have two subunits, a smaller subunit and a larger subunit. The size of these subunits is different in eukaryotes and prokaryotes.
- Eukaryotic ribosomes have a 60S larger ribosomal subunit and 40S smaller subunit. Both these subunits combine to form a single ribosome that settles at 80S.
- Prokaryotic ribosomes have a 50S larger subunit while 30S smaller subunit that combine to form 70S ribosome.
(S here stands for Svedberg; it is a unit that measures the size of particles based on their sedimentation during ultracentrifugation.)
Ribosomes are the organelles that offer the site for the interaction between mRNA and tRNA so that translation can take place. They have intrinsic enzymatic activities that assist the process of translation.
Ribosomes have three sites for tRNA.
- E (exit or empty) site, contains the tRNA that has donated its amino acid and now ready to leave the complex.
- P (polypeptide) site, contains the tRNA to which the growing polypeptide chain is attached.
- A (acceptor) site, accepts the new coming tRNA with the next amino acid to be inserted in the chain.
These sites are organized in such a way that one genetic codon is exposed to tRNA at one site.
Steps in Protein Synthesis
Just like the process of transcription, the translation process also consists of three major steps; initiation, elongation, and termination. A brief detail of these steps is as follows.
The process of translation begins with the assembly of the translational machinery. A specific codon, called initiation codon, is located at the beginning of mRNA. It signals the ribosomes to start the process of translation. The initiation codon (AUG) codes for methionine amino acid.
A molecule of mRNA is recognized by a small ribosomal subunit via a specific nucleotide sequence located upstream the initiation codon. In prokaryotes, a specific purine-rich sequence is upstream the initiation codon called Shine-Dalgarno sequence. However, this sequence is absent in eukaryotes. The eukaryotic ribosomes recognize mRNA through its keep located several nucleotides upstream the initiation codon.
The small ribosomal subunit recognizes and binds to the mRNA and begins the screening process till it reaches the initiation codon.
The initiation codon is exposed at the P site of the ribosome. This AUG code is recognized by a special tRNA, called initiator tRNA, carrying the methionine amino acid. This is the only tRNA that directly goes to the P site of ribosomes.
Once the initiator tRNA has attached, the larger ribosomal subunit joins the complex resulting in the formation of a fully functional ribosome, ready to carry out protein synthesis.
The elongation process involves the addition of new amino acids at the carboxylic end of the polypeptide chain. The next codon is exposed at the A site of the ribosome. The tRNA having corresponding amino acid recognizes this codon and inserts it into the A site.
The ribosome now has two tRNA, one at the A site and the other at the P site. A peptide bond is formed between the two amino acids. This bond formation is catalyzed by peptidyl transferase activity of larger ribosomal subunit.
As a result, a dipeptide is attached to the tRNA at the A site while the tRNA at the P site is empty.
The ribosome then translocate three nucleotides ahead on the mRNA towards its 3’ end. A new codon is exposed at the A site, the empty tRNA relocates to the P site and the tRNA with dipeptide to the P site.
The empty tRNA leaves the complex and the elongation process continues.
Just like the initiation code, there are termination codons. When the ribosome reaches one of these codons, the translation process halts. No ore tRNA comes to the A site.
Certain release factors bind to the ribosomes. These factors hydrolyze the bond between the newly constructed polypeptide and the tRNA at the P site. The polypeptide leaves the complex to undergo post-translational modifications. The other components of the complex are recycled for more protein synthesis.
The process of translation is facilitated by certain factors called translation factors. These compounds are proteins and are also made in the cytoplasm. They are of three types;
- Initiation factors, help to recognize the initiation codon and form the initiation complex
- Elongation factors, assist in the process of elongation
- Release factors, cause the release of polypeptide from the complex
The process of translation uses energy in the form of GTP (ATP is also used in eukaryotes). The translation factors also play an important role in the energy process.
One ribosome attached to one mRNA is called a monosome. There could be multiple ribosomes attached to one molecule of mRNA if multiple copies of one polypeptide are needed. The complex formed by multiple ribosomes attached to one mRNA is called polysome.
The process of gene expression complete with the synthesis of proteins. The genetic information in the gene has been expressed in the form of newly formed polypeptide. This polypeptide can serve several functions depending on its amino acid sequence, which is in turn decided by the gene.
The genetic information present in a gene is expressed in the terms of protein made by it. This process is called gene expression and involves two phases, transcription and translation.
Transcription is the process by which a transcript of the gene is made in the form of mRNA. It serves as the working copy of the gene. Other types of RNA are also made from DNA by the transcription process. It involves three steps; initiation, elongation, and termination.
The RNA molecules thus formed undergo several post-transcriptional modifications so that they become fully functional.
The second step of gene expression involves the interaction between different types of RNA to form proteins. The mRNA molecule contains a series of codons which decide the sequence of amino acids in a polypeptide. These codons are read by tRNA which carries an amino acid at its 3’end. The amino acid attached to the tRNA is inserted into the polypeptide chain.
This interaction between mRNA and tRNA occurs on the surface of ribosomes. The polypeptide thus formed undergoes modifications before becoming a fully functional protein.
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