RNA Protein Synthesis Process And Structure: Step By Step

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Protein synthesis is an elaborate process starting from the nucleus and ending in the cytoplasm.

Protein synthesis is referred to the process of producing new peptide molecules and then joining them together to produce proteins by coding genes into mRNA and then translating it.

In prokaryotes, the process involves fewer changes due to the simplicity of the DNA, but as it comes to eukaryotes the process becomes more complex.

Step by step relay of RNA protein synthesis Process and structure:

RNA  Protein synthesis process involves a total of 4 main steps:

Transcription of genes to mRNA or hnRNA:

The initial stage in decoding a cell’s genetic information is transcription. RNA polymerases are the specific enzymes used to produce RNA molecules that are complementary to each nitrogen base present on the template strand of the DNA double helix during transcription.

DNA comprises two complementary polynucleotide strands that are kept together by hydrogen bonds between base pairs in an antiparallel double helix structure. The helicase enzyme then plays its role by breaking the hydrogen bonds causing a specific region of the DNA double helix to start unwinding. As this occurs the two strands are separated and the nitrogen bases are exposed to be transcribed as codons.

330px Unwound DNA Duplex
Image depicting how a double-stranded DNA molecule unwinds
Image: Wikipedia

Even though a DNA molecule is two-stranded, only one of the strands serves as a template for hnRNA synthesis, which we simply refer to as the template strand. The coding strand is the second DNA strand complementary to the template strand as the transcripted hnRNA is identical to the coding strand, only replacing T with U.

Both DNA and RNA have an intrinsic directional sense which is biologically set in nature in how they wind or unwind, meaning there are two distinct ends of the molecule. Since all the nucleotide subunits are actually asymmetrical, due to the placement of a phosphate group on one side of the pentose sugar and the N-base on the other. This very arrangement gives rise to the directional property of the nucleic acid strands.

rna-protein-syntheis-process
The process of RNA transcription
Image: Wikipedia

The numbering of the five carbons in the pentose sugar is from 1’to 5′. The two nucleotides on the complementary strand are joined together in a special chemical bond called phosphodiester bonds. The two strands in a DNA helix are antiparallel, i.e., one runs from 3′ TO 5′; while the other runs in the opposite direction from 5′ to 3′.

Post-transcriptional processing:

It is a set of biological modifications used to convert hnRNA to mRNA. It consists of a few steps including:

  • Capping at the 5’ end:

 Capping is nothing but the attachment of a 7-methylguanosine (m7G) molecule to the 5′ end of the hnRNA molecule. In the process of capping the terminally located phosphate at the 5′ end has to be removed and a phosphatase enzyme does this. The process is then catalysed by the enzyme guanosyl transferase, which forms the diphosphate 5′ end.

This very 5′ end then reacts with a GTP molecule having three phosphate groups. It goes and attaches itself to the guanine reside by attacking and removing the alpha phosphorous atom of the GTP molecule.

The enzyme (guanine-N7-)-methyltransferase (“cap MTase”) adds a methyl group to the guanine ring from S-adenosyl methionine.

 A cap 0 structure is defined as a cap with only the (m7G) in place. The next nucleotide’s ribose can likewise be methylated to produce a cap 1. CaNucleotide methylation downstream of the RNA molecule results in cap 2, cap 3, and other structures. During this time the methyl groups go ahead and attach themselves to the 2’ oH groups of the ribose sugars. The cap protects the parent RNA transcript’s 5′ end from ribonucleases that are specialised for the 3’5′ phosphodiester linkages.

  • Tailing at the 3’ end:

The tailing of hnRNA indicates the addition of nearly 250 adenine residues to the 3’ end of the hnRNA after cleaving it. This gives rise to something scientists refer to as a poly(A) tail. The cleavage and adenylation(or addition of multiple adenine residues) only occur when a specific signal sequence is found on the 3′ end of the hnRNA. This is called a polyadenylation signal sequence (5′-AAUAAA-3′) and needs to be followed by another sequence (5′-CA-3′), which indicated the cleavage site.

Only when this specific sequence is satisfied does the cleavage and adenylation begin. Using ATP as a precursor, Poly(A) polymerase adds around 200 adenine units to the new 3′ end of the RNA molecule. The poly(A) tail binds numerous copies of poly(A)-binding protein as generated. This is to protect the 3’end of the mRNA from being digestion by ribonuclease enzyme complexes such as the CCR4-Not complex.

  • Splicing:

RNA splicing refers to the removal of introns (RNA sections that do not code for proteins) from pre-mRNA and connecting the remaining exons to form a single uninterrupted molecule. Exons are mRNA segments that are “translated” or converted into proteins. They are the mRNA molecule’s coding segments. Even though most RNA splicing occurs after the pre-mRNA has been fully produced and end-capped, transcripts with a large number of exons can be spliced co-transcriptionally.

 A huge protein complex termed the spliceosome catalyzes the splicing reaction, which is made up of proteins and tiny nuclear RNA molecules that recognize splice sites in the hnRNA sequence. Many hn-mRNAs, such as those coding antibodies, can all be spliced in a variety of ways to yield various mature mRNAs encoding different protein sequences. This is known called alternative splicing, and it allows for the creation of a wide range of proteins from a small amount of DNA.

Translation of mRNA to protein:

The mRNA is converted or in scientific jargon “translated”  to a chain of amino acids according to the genetic code present on the coding strand of the  DNA. This is how the DNA sequence relates to the amino acid sequence in the polypeptide chain which is the second primary step in gene expression during translation. Each codon in mRNA comprises three Nitrogenous bases, and each codon indicates or codes a particular amino acid, or some to start or stop the process of translation. The mRNA sequence is thus employed as a template to construct the chain amino acid chain that forms a protein in the correct order. mRNA translation occurs in a series of steps as mentioned below in detail:

800px Protein synthesis.svg
RNA translation to protein
Image: Wikipedia
  • Initiation:

The ribosome comes and surrounds the mRNA of interest to be translated and then comes and attaches to the start codon.  The start codon does not code for any amino acid but rather only acts as the site of attachment of the ribosomes starting the process of translation.

  • Elongation:

Elongation includes the final tRNA recognized by the smaller ribosomal subunit to carry the amino acid and transfer it to the larger ribosomal subunit. This larger subunit then attached this amino acid to the previously recognized and admitted tRNA molecule. These 2 simultaneous steps are called accommodation and transpeptidation. The ribosome then moves to the next codon and continues the process in the same way, called translocation. This continued moving of amino acids results in the formation of a large polypeptide chain, formed by amino acids joined by peptide links.

  • Termination:

When the ribosome reaches a codon sequence like UAA, UAG or UGA referred to as stop codon, it detaches from the mRNA and the polypeptide it has translated. This marks the end of the process of translation causing termination.

Post-translational modifications of the polypeptide chain translated:

The last step of RNA protein synthesis refers to the changes that the polypeptide chain undergoes before being assembled into protein macromolecules.

Post-translational modifications (PTMs) promote the functional variety of the proteome by covalently attaching functional groups or proteins, proteolytically cleaving regulatory subunits, or destroying entire proteins. These alterations to the protein molecules include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis.

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Image showing the post-translational modifications undergone by Insulin(hormone)
Image: Wikipedia

These modifications are important and play a significant role not only in normal cell health and function but also in the treatment and prevention of diseases. Identifying and comprehending PTMs is therefore crucial in the study of cell biology as well as disease treatment and prevention. Here we will discuss some of them.

  1. Phosphorylation:

Protein phosphorylation is one of the few biological reversible processes making it one of the best studies biological phenomena among scientists. It is mostly seen on amino acids like serine, threonine which have polar neutral side chains and tyrosine which has an aromatic side chain. Phosphorylation regulates several biological functions, including cell cycle, proliferation, death, and signal transduction pathways.

  • Glycosylation:

Protein glycosylation is recognised as a fundamental post-translational modification that has a considerable impact on protein folding, shape, distribution, stability, and activity. Glycosylation refers to a wide spectrum of sugar-moiety additions to proteins, from simple monosaccharide changes in nucleus transcription factors to extremely complicated branched polysaccharide changes in cell surface receptors. Many cell surface and secreted proteins contain carbohydrates in the form of asparagine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides.

  • S-nitrosylation

S-nitrosylation is a reversible process, and SNOs(S-nitrothiols) have a brief half-life in the cytoplasm due to a plethora of reducing enzymes that denitrosylate proteins, including glutathione (GSH) and thioredoxin. Due to their high reactivity, instead of free-floating in the cytoplasm, SNOs are retained in organelles like membranes, vesicles, interstitial spaces and even in lipophilic proteins so they are not simply denitrosylated.

 Caspases, which mediate apoptosis, for example, are stored as SNOs in the mitochondrial intermembrane gap.

Once extracellular or intracellular signals come through the Caspases are released into the cytoplasm. Since the cytoplasm is highly reducing in nature the proteins are rapidly denitrolysed. This denitrolysation activates the activation of the caspase and induces the process of apoptosis.

  • Ubiquitination:

Ubiquitination is the attachment of  Ubiquitin to protein, an 8-kDa polypeptide made up of 76 amino acids that attach to the Î-NH2 of lysine in protein targets via ubiquitin’s C-terminal glycine. After the first monoubiquitination event, a ubiquitin polymer may form, and polyubiquitinated proteins are subsequently identified by the 26S proteasome, which catalyses ubiquitin breakdown and ubiquitin recycling. The following experiment demonstrates a method for detecting ubiquitinated proteins.

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