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MECHANISM OF PROTEIN SYNTHESIS

MECHANISM OF PROTEIN SYNTHESIS

The protein synthesis occurs in two steps: Transcription and Translation.

Transcription

The process of synthesis of RNAs (mRNA, tRNA and rRNA) from DNA by the enzyme RNA polymerase is known as transcription. At the time of transcription, the RNA polymerase binds with double stranded DNA (gene) at a particular site (in prokaryotes known as promoter site) and after unwinding of the two strands of DNA by the rotation of the DNA, it starts copying one of
the two strands, known as coding strand (sense strand or template strand). The other strand of the DNA, which is not copied for the RNA synthesis, is known as non-coding strand (antisense strand) (Fig. 8.6).

Fig.8.6

Fig. 8.6 Transcription in prokaryotes.

In eukaryotes there are four types of RNA polymerases for the synthesis of different types of RNA molecules:

 
i.RNA polymerase I (or A) found in nucleolus for the synthesis of rRNAs (28S and 18S).

ii.RNA polymerase II (or B) found in nucleoplasm for the synthesis of HnRNA (heterogeneous nuclear RNA) which, after processing gives rise to mRNA.

iii.RNA polymerase III (C) found in nucleoplasm for the synthesis of tRNA and 5S rRNA.

iv.Organeller RNA polymerase found in organelles, mitochondria and chloroplasts, (in photosynthetic organisms) and resembles the prokaryotic RNA polymerase.

 

In prokaryotes, only one type of RNA polymerase synthesizes all the types of RNAs. It is made up of 5 subunits (proteins), where α2ββ’ makes the core enzyme that is found loosely attached
with a σ factor. The σ factor helps in the recognition of promoter site and after initiating the transcription, it gets dissociated from the holoenzyme (σσ2ββ). Now, the RNA is synthesized by the movement of core enzyme (σ2ββ) along the DNA.

Fig.8.7

Fig.8.7 Hairpin loop formation in mRNA.

Fig.8.8

Fig. 8.8 Mechanism of transcription in prokaryotes

The termination of transcription occurs due to the presence of certain terminator sites on DNA. In prokaryotes, the termination is mainly done by in two ways, which involves the presence of certain palindromic sequences (Inverted repeats with the same sequences on the two strands of DNA when read in 5’→ 3′ direction. Thus, the presence of complementarity of bases on the same strand causing loop formation in mRNA due to intrastrand complementary base pairing) on DNA near the termination signals (Fig. 8.7 and 8.8).

 i.ρ-dependent Termination: In some prokaryotes, the termination of transcription is helped by a ρ (rho) protein that gets attached at the 5′ end of the newly synthesizing mRNA. The ρ then moves along the mRNA and induces the formation of hairpin loop near the 3′ end of mRNA due to the presence of inverted repeated sequences. This helps in the detachment of mRNA from the DNA (Fig. 8.9).
Fig.8.9

Fig. 8.9 ρ – dependent termination of transcription in prokaryotes.

 

i. ρ-independent Termination: In many prokaryotes after the hairpin ‘loop formation at 3’ end of mRNA, a long run of U (A in coding strand of DNA) is found and due to      weaker U-A pairing in mRNA-DNA complex, the glRNA gets detached from the DNA (Fig. 8.10).

 

In prokaryotes, the transcription and translation processes occur simultaneously as there is no nuclear membrane to separate the genetic DNA and the cytoplasm.

Fig.8.10

Fig. 8.10 ρ-independent termination of transcription in prokaryotes.

 

In eukaryotes, the termination sites in DNA are present far away from the corresponding actual 3′ end of mRNA, thus, to produce HnRNA (heterogeneous nuclear RNA). The 3′ end of the mRNA is generated after the processing of HnRNA by snurp (small nuclear RNA-protein complex). In addition to these extra nucleotides at 3′ end, the HnRNA may also contain extra nucleotide sequences at the 5′ end and at the internal positions. These extra nucleotide sequences at internal positions are called introns, whereas, the nucleotide sequences in between the introns

Fig.8.11

Fig. 8.11 The eukaryotic gene (above) and mRNA (below).

that are present in mRNA and contain the information of proteins are known as exons. Thus, HnRNA produced after transcription is quite longer than the mRNA. Most of the extra nucleotide sequences, including introns, are cleaved by snurp. Moreover, after removal of the extra nucleotides from the 3′ end of the HnRNA, poly A tail is added that is required for the stability of the mRNA. Similarly, after removal of extra nucleotides from the 5′ end, a cap of 7-methyl guanosine (7mG) is added that is required for the translation process. The production and processing of HnRNA occurs in the nucleus from where it escapes into the cytoplasm through nuclear pores for the translation process (Fig 8.11 and 8.12).

Fig.8.12

Fig.8.12

Fig. 8.12 Transcription and processing of mRNA in eukaryotes,

Translation

The process of synthesis of proteins from mRNA (translation of language of nucleic acids into the language of proteins) is called translation. There are 20 different types of amino acids, which constitute various proteins, and these amino acids themselves cannot recognize their respective codons in the mRNA. Different amino acids are carried by their specific tRNA molecules at the
site of protein synthesis (mRNA). There are about 55 types of tRNA molecules available in the cytoplasm, so that one amino acid may have more than one tRNAs.

Various steps for the translation process in prokaryotes arc:

 

  1. The binding of an amino acid at the 3′ end of its specific tRNA is facilitated by an enzyme, amino acyl-tRNA synthetase, that is also specific for a particular amino acid.
  2. The initiation codon AUG (rarely GUG) is always occupied by the amino acid, formyl methionine (formylation of methionine is done by the enzyme translormylasc) which is
    carried by its specific tRNAfmet by codon-anticodon pairing (in intCI’IlHI positions, methionine is carried by tRNA met). (In eukaryotes the initiation of translat ion is done by the amino acid, methionine) (Fig. 8.13 and 8.14)
  3. Fig.8.1314.jpg

    Fig 8.13 Initiation of translation in prokaryotes

    3. Initiation factor 3 (IF3) helps in the dissociation of70S ribosome into 30S and 50S subunits. Now, the IF3 attached with 30S subunit, binds at the 5′ end of the mRNA. (In eukaryotes the initiation codon lies far away from the 5′ end of mRNA and the smaller subunit(40S) of ribosome moves along the mRNA to find the initiation codon).

  4. Fig.8.14
    Fig.8.14
    Fig 8.14 Initiation of translation in eukaryotes.
    4. The ribosomes have two sites, the A-site (amino acyl site) for the location of new amino acyl-tRNA (except for the fmet-tRNAfmet) and a P-site (peptidyl site) for locating the tRNA attached with newly synthesizing amino acid chain (di and polypeptide). The fmet-tRNAfmet after binding withIF2, is located on the initiation codon AUG at the P-site of the 30S subunit of ribosome which requires energy in the form of GTP. The role of IF1 is not
    known. (In eukaryotes more initiation factors i.e., eIF1 to e1F6, are involved in nitiation).

    5. After initiation of the translation process, elongation process for the formation of dipeptide and ultimately the polypeptide chain is started by the attachment of AA2-tRNAAA2 (amino acyl tRNA or second amino acid attached with its specific tRNA) at its respective codon at the empty A-site with the help of the elongation factors EFTu (temperature unstable elongation factor) and EFTs (temperature stable elongation factor). This process also requires GTP. (In eukaryotes the elongation factors eEF1 and eEF2 are involved in the elongation process).

    6. Now, the enzyme peptidyl transferase helps in the formation of peptide bond at the A-site between the -COOH group of AA2-tRNAAA2 (peptidyl tRNA for second and subsequent rounds of elongations) and -NH2 group of fmet (thereafter, between 2nd and subsequent amino acids) (Fig. 8.15).

    7. Thereafter, the naked tRNA at the P-site is removed with the help of the elongation factor EFG and the energy GTP as the ribosome moves by 3 nucleotides along the mRNA in 5’→ 3′ direction. Now, the P-site carries the fmet-AA2-tRNAAA2 (tRNA attached with a dipeptide).

    8. The AA3-tRNAAA3 (3rd amino acid attached with its tRNA) occupies the empty A-site. Thus this process of movement of ribosome along the mRNA and entry of new
    AAntRNAAAn, at the A-site is repeated.

    9. As the ribosome moves by two codons, next round of protein synthesis is initiated by the attachment of a new ribosome. Thus, at a time, a single mRNA is found to be attached with many ribosomes with their polypeptides of different length, (shortest polypeptide at the 5′ end of the mRNA and longest at the 3′ end), called polysomes.
    Fig.8.15
    Fig.8.15
    Fig. 8.15 Peptide bond formation in growing polypeptide.
    10. Ultimately, the A-site of ribosome is occupied by the termination codon (UAA,UAG or UGA) at the 3′ end of mRNA, which is not recognized by any tRNA. Thus, the termination of the protein synthesis is helped by the release factors RFl, RF2 and RF3 (in eukaryotes eRF1), which release the newly synthesized polypeptide chain from the P-site (Fig. 8.16).
    Fig.8.16

    Fig. 8.16 Translation process of protein synthesis in prokaryotes.

    11. The newly synthesized polypeptide is modified by the removal of formyl group of the first amino acid, formyl-methionine. Moreover, a few amino acids from the N-terminal (-NH2 terminal) or from the C-terminal (- COOH terminal) or from both the termini may be removed, The primary structure of the polypeptide gets folded variously to produce three-dimensional secondary and tertiary structures.