Protein Synthesis | Transcription phase | Translation phase |

Protein Synthesis

The cell preserves hereditary information in genes, which work as instructions for building proteins. The gene instructions from the DNA transfer are stored in the molecules of ribonucleic acid, the "mRNA," and then the mRNA transcript translates the instructions for the synthesis of protein. This one flow of hereditary information from DNA to mRNA and from mRNA to protein is known as the "center dogma of life."

There are three different forms of RNA in cells, each of which has a different function: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). All three types of RNAs are essential for protein synthesis. The process of protein synthesis involves two stages known as transcription and translation. The process of transcription and translation is discussed below.

mRNA Transcription 

Transcription involves the transfer of information from a gene in DNA into an mRNA molecule. In eukaryotic organisms, transcription occurs inside the nucleus, while in prokaryotic organisms, it takes place in the cytoplasm. It is defined as

"The formation of RNA on a template of a DNA strand is called transcription."

Transcription includes the following steps:

Template binding

Transcription begins when an enzyme called RNA polymerase binds to the transcription start site of a gene on a region of DNA called a promoter. A promoter is a specific sequence of DNA that is important for the recognition of RNA polymerase, known as the transcription start site. It is known as the "TATA box" in eukaryotes because it is rich in thymine and adenine nitrogenous bases. RNA polymerase cannot initiate transcription on its own until the transcription factors recognize the sequence and help the RNA polymerase in their search for promoters to start the transcription. After RNA polymerase binds to a promoter, the enzyme starts to unwind and separate the two strands of the double helix, exposing the DNA's nitrogen-containing bases. The enzyme recognizes the nitrogen base and adds the complementary base on the growing chain, the two bases join each other by a phosphodiester bond and initiate the formation of the mRNA chain. DNA bases act as a template for the formation of the mRNA molecule. In any particular transcription, however, only one of the two strands of DNA is transcribed; this strand is called the template strand or antisense strand, while the opposite strand is called the coding sense or sense strand. Thus, the resulting mRNA is complementary to the DNA template from which it is formed.

Chain elongation

RNA polymerase separates apart the double helix and moves downstream along the DNA template strand from the initiation site, always in the 3’ to 5’ direction. While the growing mRNA always synthesize from 5’ to 3’ direction. The enzyme reads each nucleotide and adds it on complementary mRNA. When the RNA nucleotides are added, they are linked together with sugar-to-phosphate covalent bonds. In eukaryotic cells, the RNA nucleotides are found in the nucleus; in prokaryotic cells, they are in the cytoplasm. As the transcription grows downstream, the two strands of DNA close up by forming hydrogen bonds between complementary bases and reform the double helix.

Chain termination

RNA polymerase continues to elongate the mRNA until it reaches the termination site. It is the specific sequence of nucleotides that signals the termination or end of the mRNA transcription. The mRNA, the transcript of a gene, is released, and polymerase subsequently dissociates from the DNA.

In prokaryotes, the mRNA is directly released into the cytoplasm, where it is translated into protein. In eukaryotes, mRNA has to travel a long distance from the nucleus to the cytoplasm; therefore, mRNA must be processed before it is released.

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Figure 1: Labeled diagram of transcription. 

mRNA processing in eukaryotes

Before being released into the cytoplasm for protein synthesis, eukaryotic mRNA undergoes post-transcriptional modification, which includes 5' capping, mRNA splicing, and polyadenylation.

Addition  of 5 ’capping on mRNA:  

Before the release enzyme called guanyl transferase adds the cap, it is in the form of 7-methyl GTP (7-methyle-guanosine-5'-triphosphate) on the first nucleotide of mRNA at 5’. It is a highly methylated modified guanine base. The 5’ cap protects the mRNA from degradation, prevents it from being attacked by exonucleases, and even assists other post transcriptional events, including splicing, polyadenylation, and recruitment of translational machinery.

mRNA splicing

The growing mRNA stretch contain both intron (a noncoding region) and exon (a coding region), and the pre-mRNA undergoes alternative splicing, which leads to different combinations of mRNA before release. The different mRNAs produce different proteins.

Polyadenylation

The tail is in the form of poly A linked to 3’ end of mRNA by the help of an enzyme called poly-A polymerases. Poly-A tail is in fact a stretch of adenosine monophosphate, which contains almost 100 to 250 nucleotide residues. The newly synthesized mRNA contains a 3’ UTR region that is enriched with AU nucleotides; the 3’ UTR contains a sequence AAUAAA that assists the addition of a tail. The end 3’ region is cleaved by a set of proteins to cleave the hydroxyl group, and the enzyme poly-A polymerases assist in the addition of the poly-A tail. The tail protects the mRNA from a variety of cytoplasmic enzymes, stabilises the mRNA, prevents it from degradation, and allows the export of mature mRNA from the nucleus to the cytoplasm.

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Figure 2: Structure of mature mRNA.


Translation

The equipment for translation is located in the cytoplasm, where a cell keeps its protein supply. This translation machinery includes: transfer RNA (tRNA), which is a single strand of RNA folded into a compact shape with three loops, one of the loop has a three-nucleotide sequence called an anticodon. A codon can be complementary to one of the 64 codons of the genetic code. Opposite the anticodon on a tRNA molecule is a site at which the molecule carries an amino acid. The ribosome is composed of protein and rRNA; each ribosome has two subunits. The smaller subunit binds to the mRNA, and the larger subunit has three functional sites. Two sites called P and A bind to the tRNA, while the third site catalyzes the formation of peptide bonds between amino acids in growing protein chain.

tRNA and genetic code

In the presence of an enzyme called aminoacyl-tRNA synthetases, each tRNA is chemically linked with a specific amino acid. The tRNA is covalently linked with the specific amino acid, and ATP provides the energy for the binding reaction. There are different types of tRNA, ranging from 40 to 60, depending on the type of cell and species, each bearing the specific anticodon for the 20 different amino acids floating in the cytoplasm. There are 64 different codons; multiple codons can be encoded for the same amino acid. AUG on an mRNA read is a start codon and always encodes for methionine; similarly, UAA, UAG, and UGA are stop codons and do not encode for any amino acid.

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Figure 3: Genetic codes for particular amino acids. 
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Figure 4: Labeled diagram of structure of tRNA.


Translation definition

"Translation is the process by which a protein is synthesized; the liner amino acid sequence is directed by mRNA sequences that are specified by a gene."

The mechanism of translation The process consists of the following stages.

Translation Initiation

Translation begins when mRNA binds to the smaller ribosomal subunit in the cytoplasm. The ribosomal S1 protein plays an important role in the recognition of mRNA and its binding. Similarly, the ribosome also recognizes the 7-methyle-guanosine cap and scans the 5’ UTR (untranslated region) for binding and initiation of translation. The mRNA is oriented in such a way that the start codon (a codon that signals the beginning of a protein chain) is sitting in the P site, and at the same time, the initiator tRNA with the anticodon UAC base pairs with the initiation codon AUG bearing the amino acid methionine (met) and initiates the complex. The larger ribosomal subunit joins the initiation complex, and a protein called initiator factor brings these translation components together. In a bacterial cell, almost 3 initiator factors (IF1, IF2, and IF3) are required to initiate the translation, while in eukaryotes, which are complex organisms, there are a number of initiator factors, but at least 11 are required to initiate the translation (eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4F, eIF4G, eIF4B, eIF5 and eIF5B, eIF5 and eIF2B, respectively). GTP, which is closely related to ATP, provides energy for the initiation process. The larger subunit has three sites: the P (peptidyl) site, the A (aminoacyl) site, and the E (exit) site. The initiator tRNA first binds to the P site, which later holds the nascent peptide chain, and the A site is available for the tRNA (aminoacylated) bearing the next amino acid. The E site holds the deacylated tRNA before the tRNA leaves the complex after the transfer of amino acid.

Translation Initiation Mechanism:

  • The initiation phase of translation begins when initiation factors (eIF1, eIF3, and eIF1A) bind to the smaller ribosomal units, while initiation factor eIF2 along with GTP complexes with initiator tRNA brings it to the ribosome, and a group of eIF4 initiation factors brings mRNA to the ribosome.
  • As the translation machinery was brought to the ribosome, the ribosome then scanned the mRNA downstream to identify the initiation codon AUG. This process required energy, which was provided by ATP hydrolysis.
  • As the start codon was identified, another initiation factor known as eIF5 triggered the GTP hydrolysis, as a result, the eIF2_GTP complex dissociates and tRNA is released and binds to the A site.
  • The larger subunit then binds to the complex and continues the process.
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Figure 5: Mechanism of translation initiation.

The elongation cycle of translation

After the formation of the initiation complex, the very next step is elongation of the peptide chain. As the incoming aminoacylated tRNA binds to the codon at the P site, the elongation factor and GTP are required for the process. The amino acid at P site forms a covalent link with the amino acid at A site, and a peptide is formed between the new amino acid and the growing polypeptide chain. The reaction of peptide bond formation is catalysed by peptidyl transferase (a catalytic RNA). The growing peptide chain first transferred to the tRNA at A site. The ribosome moves along the mRNA downstream from 5’ to 3’. As the ribosome translocates downstream, there is a shift of one codon forward. The initiator tRNA or tRNA at P site shifted to the E site and the tRNA bearing the polypeptide chain translocated at P site, A site is again available for the next coming tRNA.

Mechanism of chain elongation

The following are the steps involved in chain elongation:

  • The elongation phase of translation involves the elongation of polypeptide chains, as the initiator methyonyl tRNA sits at the P site and the second aminoacyl tRNA is brought to the A site by the elongation factor EF-Tu complex with GTP, having an anticodon matched exactly to the code at the A site.
  • GTP hydrolysis the bond, and GTP_EF-Tu complex leaves the ribosome and newly coming aminoacylated tRNA sits at A site.
  • A peptide bond is formed between two amino acids, which results in the transfer of methionine (an amino acid at the P site) to the amino acid at the A site.
  • After the transfer, the ribosome is translocated three nucleotides forward; this process is conducted by the elongation factor EF-Gu.
  • As the ribosome translocate the deaminoacylated tRNA shifts at E site, the tRNA at A site bearing the peptide chain shifts at P site and A site sets free for incoming new aminoacylated tRNA.
  • The process is to continue until the whole mRNA is fully decoded and reaches to the end.
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Figure 6: Mechanism of translation elogation.

Termination of translation

When a ribosome reaches a termination codon on a strand of mRNA, a protein called release factor recognizes the stop codon and binds to the A site instead of tRNA. In eukaryotes, a single release factor, eRF1, recognizes all three stop codons, whereas prokaryotes have two release factors.RF1: It can recognize UGA and UUA, and RF2: It can recognize UUA and UGA. The release factor hydrolyzes the bond between the tRNA in the P-site and the last amino acid of the polypeptide chain. Both polypeptide and tRNA are then free to depart from the ribosome, and the two ribosomal units dissociate from the mRNA, and that’s how the translation ends and the process of protein synthesis is completed. However, one mRNA can be translated by several ribosomes at the same time.

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Figure 7: Mechanism of translation termination.


Post translational modification

Protein could not be synthesize or fully functional soon after the translation. As the translation ends, the nascent polypeptide chain undergoes posttranslational modification. These modifications include the removal of Met from certain proteins and the addition of functional groups that include methyl, phosphate, acetate, and amide. Lipids and sugar are also added to certain proteins. Other proteins undergo conformational changes, breakage of some bonds, formation of new covalent linkages, excision of some peptide chains, and reorientation of peptide chains. All these posttranslational changes lead to the final protein product, which is then available to perform its function.

Sources

Clark, D. P., & Pazdernik, N. (2012). Molecular biology. Elsevier
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