PROTEIN SYNTHESIS

INTRODUCTION – PROTEIN SYNTHESIS

• Proteins are the end products of most information pathways. A typical cell requires thousands of different proteins at any given moment. These must be synthesized in response to the cell’s current needs, transported (targeted) to their appropriate cellular locations, and degraded when no longer needed.
• An understanding of protein synthesis, the most complex biosynthetic process, has been one of the greatest challenges in biochemistry. Eukaryotic protein synthesis requires more than 70 different ribosomal proteins; 20 or more enzymes to activate the amino acid precursors; a dozen or more auxiliary enzymes and other protein factors for the initiation, elongation, and termination of polypeptides; perhaps 100 additional enzymes for the final processing of different proteins; and 40 or more kinds of transfer and ribosomal RNAs. Overall, almost 300 different macromolecules cooperate to synthesize polypeptides.
• Many of these macromolecules are among the most abundant to be found in any cell. Some are organized into the complex three dimensional structure of the ribosome.
•  To appreciate the central importance of protein synthesis, consider the cellular resources devoted to this process.
• Protein synthesis can account for up to 90% of the chemical energy used by a cell for all biosynthetic reactions. Every bacterial, archaeal, and eukaryotic cell contains from several to thousands of copies of many different proteins and RNAs. The 15,000 ribosomes, 100,000 molecules of protein synthesis–related protein factors and enzymes, and 200,000 tRNA molecules in a typical bacterial cell can account for more than 35% of the cell’s dry weight.

THE GENETIC CODE

Francis Crick’s provided the reasoning on how the genetic information encoded in the 4-letter language of nucleic acids could be translated into the 20-letter language of proteins. A small nucleic acid (perhaps an RNA) could serve the role of an adaptor, with one part of the adaptor molecule binding a specific amino acid and another part recognizing the nucleotide sequence encoding that amino acid in an mRNA. This idea was soon verified.

The tRNA adaptor, the same molecule that activates the amino acid for peptide bond formation, also “translates” the nucleotide sequence of an mRNA into the amino acid sequence of a polypeptide. The overall process of mRNA-guided protein synthesis is often referred to simply as translation.

With four possible nucleotides at each position, the total number of permutations of these triplets is 64 (4 multiply by 4 multiply by 4), a value well in excess of the number of amino acids. Which of these triplet codons are responsible for specifying which amino acids,will form from which triplet codon, with the remaining three triplets being chain terminating signals. This means that many amino acids are specified by more than one codon, a phenomenon called degeneracy.

Codons specifying the same amino acid are synonyms. For example, UUU and UUC are synonyms for phenylalanine, whereas serine is encoded by the synonyms UCU, UCC, UCA, UCG, AGU, and AGC

PROPERTIES

• The group of nucleotide that specify one amino acid is a codon.
• The gentic code consist of 64 codon,61 sense codon that code for 20 amino acid and 3 stop codon
• The genetic code is unambiguous meaning that each triplet specify only a single amino acid.
• No punctuations is used in codon.
• The code is degenerate, meaning that a given amino acid can be specified by more than one amino acid.
• Genetic code is nonoverlapping means that no single base can take part in formation of more than one codon.
• It is usual to describe the genetic code as universal code.

THE WOBBLES HYPOTHESIS

It states that the base at the 5 end of the anticodon is not as spatially confined as the other two, allowing it to form hydrogen bonds with any of several bases located at the 30 end of a codon. Not all combinations are possible, with pairing restricted to those shown in  For example, U at the wobble position can pair with either adenine or guanine, while I can pair with U, C, or A.

The pairings permitted by the wobble rules are those that give ribose– ribose distances close to that of the standard A:U or G:C base pairs. Purine–purine (with the exception of I:A pairs) or pyrimidine–pyrimidine pairs would give ribose– ribose distances that are too long or too short, respectively. The wobble rules do not permit any single tRNA molecule to recognize four different codons. Three codons can be recognized only when inosine occupies the first (50 ) position of the anticodon.

MESSENGER RNA

The protein-coding region(s) of each mRNA is composed of a contiguous, non-overlapping string of codons called an open reading frame (commonly known as an ORF). Each ORF specifies a single protein and starts and ends at internal sites within the mRNA. That is, the ends of an ORF are distinct from the ends of the mRNA. Translation starts at the 5prime  end of the ORF and proceeds one codon at a time to the 3prime end.

The first and last codons of an ORF are known as the start and stop codons. In bacteria, the start codon is usually 5prime -AUG-3prime, but 5prime-GUG-3prime and sometimes even 5prime -UUG-3prime are also used. Eukaryotic cells always use 5prime -AUG-3prime as the start codon.

Stop codons, of which there are three (5prime -UAG-3prime , 5 prime -UGA-3prime , and 5prime -UAA-3prime ), define the end of the ORF and signal termination of polypeptide synthesis. It is a contiguous stretch of codons “read” in a particular frame (as set by the first codon) that is “open” to translation because it lacks a stop codon (i.e., until the last codon in the ORF). mRNAs contain at least one ORF. The number of ORFs per mRNA is different between eukaryotes and prokaryotes.

Eukaryotic mRNAs almost always contain a single ORF. In contrast, prokaryotic mRNAs frequently contain two or more ORFs and hence can encode multiple polypeptide chains. mRNAs containing multiple ORFs are known as polycistronic mRNAs, and those encoding a single ORF are known as monocistronic mRNAs.

To facilitate binding by a ribosome, many prokaryotic ORFs contain a short sequence upstream (on the 5prime  side) of the start codon called the ribosomebinding site (RBS). This element is also referred to as a Shine –Dalgarno sequence.

Eukaryotic mRNAs recruit ribosomes using a specific chemical modification called the 5prime cap, which is located at the extreme 5prime  end of the RNA

TRANSFER RNA

• tRNA molecules to which an amino acid is attached are said to be charged, and tRNAs that lack an amino acid are said to be uncharged. Charging requires an acyl linkage between the carboxyl group of the amino acid and the 2prime- or 3prime-hydroxyl group  of the adenosine nucleotide that protrudes from the acceptor stem at the 3prime end of the RNA.
• All aminoacyl-tRNA synthetases attach an amino acid to a tRNA in two enzymatic steps.
•  Step one is adenylylation in which the amino acid reacts with ATP to become adenylylated with the concomitant release of pyrophosphate. Adenylylation refers to transfer of AMP, as opposed to adenylation,
•  Step two is tRNA charging in which the adenylylated amino acid, which remains tightly bound to the synthetase, reacts with tRNA. This reaction results in the transfer of the amino acid to the 3prime end of the tRNA via the 2 prime – or 3 prime -hydroxyl and the release of AMP.

ACTIVATION OF AMINO ACID

• Attachment of an amino acid to tRNA  involves covalent linkage between the carboxyl group of amino acid and the 2 prime or 3 prime hydroxyl group of adenine containing nucleotide. This process is catalyzed by an enzyme called aminoacyl- tRNA  synthetase. When a  tRNA  is charged with amino acid corresponding to its anticodon it is called aminoacyl- tRNA.
• The aminoacylationis performed by two steps in which amino acid is first activated by ATP forming an intermediate an aminoacyl adenylate  and in the second step the amino acid is transferred to 3 prime end of tRNA

PROCESS OF TRANSLATION

INITIATION

• In step 1 , the 30S ribosomal subunit binds two initiation factors, IF1 and IF3. Factor IF3 prevents the 30S and 5 prime  subunits from combining prematurely. The mRNA then binds to the 30S subunit. The initiating (5′)AUG is guided to its correct position by the Shine-Dalgarno sequence in the mRNA. This consensus sequence is an initiation signal of four to nine purine residues, 8 to 13 bp to the 5′ side of the initiation codon.
• The sequence base-pairs with a complementary pyrimidine rich sequence near the 3′ end of the 16S rRNA of the 30S ribosomal subunit. This mRNA-rRNA interaction positions the initiating (5′)AUG sequence of the mRNA in the precise position on the 30S subunit where it is required for initiation of translation. The particular (5′)AUG where fMettRNAfMet is to be bound is distinguished from other methionine codons by its proximity to the Shine-Dalgarno sequence in the mRNA.
•  Bacterial ribosomes have three sites that bind tRNAs, the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site. The A and P sites bind aminoacyl-tRNAs, whereas the E site binds only uncharged tRNAs that have completed their task on the ribosome. Both the 30S and the 50S subunits contribute to the characteristics of the A and P sites, whereas the E site is largely confined to the 50S subunit.
• The initiating (5′)AUG is positioned at the P site, the only site to which fMet-tRNAfMet can bind. The fMet-tRNAfMet is the only aminoacyl-tRNA that binds first to the P site; during the subsequent elongation stage, all other incoming aminoacyl-tRNAs (including the Met-tRNAMet that binds to interior AUG codons) bind first to the A site and only subsequently to the P and E sites. The E site is the site from which the “uncharged” tRNAs leave during elongation. Factor IF1 binds at the A site and prevents tRNA binding at this site during initiation.
• In step 3 , this large complex combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF2 is hydrolyzed to GDP and Pi , which are released from the complex. All three initiation factors leave the ribosome at this point. Completion of the steps in produces a functional 70S ribosome called the initiation complex, containing the mRNA and the initiating fMet-tRNAfMet .
• The correct binding of the fMet-tRNAfMet to the P site in the complete 70S initiation complex is ensured by at least three points of recognition and attachment: the codon-anticodon interaction involving the initiation AUG fixed in the P site, the interaction between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA, and the binding interactions between the ribosomal P site and the fMet-tRNAfMet . The initiation complex is now ready for elongation.

ELONGATION

Elongation requires

1. The initiation complex described above
2. aminoacyl-tRNAs
3. a set of three soluble cytosolic proteins called elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria)
4. GTP. Cells use three steps to add each amino acid residue, and the steps are repeated as many times as there are residues to be added.

In the first step of the elongation cycle, the appropriate incoming aminoacyl-tRNA binds to a complex of GTP-bound EF-Tu. The resulting aminoacyl-tRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex. The GTP is hydrolyzed and an EF-Tu–GDP complex is released from the 70S ribosome. The EF-Tu–GTP complex is regenerated in a process requiring EF-Ts and GTP.

Enzyme peptidyl transferase catalyse the formation of peptide bond between two amino acid attached with two separate tRNAs present at P and A site first peptide bond  formation occur when the formylated methionine. Carried by the tRNA at the P site is transferred to amino acid carried by the aminoacyl – tRNA at A site, transfer of the amino acid from P site to A site generates a deacylated tRNA (lacking any amino acid) lies in P site and growing peptide chain contain  peptidyl-tRNA is in the A site. The deacylated tRNA leaves the ribosome via F site.

In the final step of the elongation cycle, translocation, the ribosome moves one codon toward the 3′ end of the mRNA . This movement shifts the anticodon of the dipeptidyl-tRNA, which is still attached to the second codon of the mRNA, from the A site to the P site, and shifts the deacylated tRNA from the P site to the E site, from where the tRNA is released into the cytosol.

The third codon of the mRNA now lies in the A site and the second codon in the P site. Movement of the ribosome along the mRNA requires EF-G (also known as translocase) and the energy provided by hydrolysis of another molecule of GTP. A change in the three-dimensional conformation of the entire ribosome results in its movement along the mRNA. Because the structure of EF-G mimics the structure of the EF-Tu–tRNA complex, EF-G can bind the A site and, presumably, displace the peptidyl-tRNA.

After translocation, the ribosome, with its attached dipeptidyl-tRNA and mRNA, is ready for the next elongation cycle and attachment of a third amino acid residue. This process occurs in the same way as addition of the second residue. For each amino acid residue correctly added to the growing polypeptide, two GTPs are hydrolyzed to GDP and Pi as the ribosome moves from codon to codon along the mRNA toward the 3′ end.

TERMINATION

Elongation continues until the ribosome adds the last amino acid coded by the mRNA. Termination, the fourth stage of polypeptide synthesis, is signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA), immediately following the final coded amino acid. Mutations in a tRNA anticodon that allow an amino acid to be inserted at a termination codon are generally deleterious to the cell. In bacteria, once a termination codon occupies the ribosomal A site, three termination factors, or release factors—the proteins RF1, RF2, and RF3— contribute to

1. hydrolysis of the terminal peptidyl-tRNA bond
2. release of the free polypeptide and the last tRNA, now uncharged, from the P site
3. dissociation of the 70S ribosome into its 30S and 50S subunits, ready to start a new cycle of polypeptide synthesis.

 RF1 recognizes the termination codons UAG and UAA, and RF2 recognizes UGA and UAA. Either RF1 or RF2 (depending on which codon is present) binds at a termination codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid. The release factors have domains thought to mimic the structure of tRNA, as shown for the elongation factor EF-G. The specific function of RF3 has not been firmly established, although it is thought to release the ribosomal subunit. In eukaryotes, a single release factor, eRF, recognizes all three termination codons.

RIBOSOMAL RESCUE

Ribosomes may stall during protein biosynthesis, especially while translating an mRNA that is damaged or incomplete. When the ribosome encounters the end of an mRNA before encountering a stop codon, the translocation step leads to formation of a stable “non-stop complex,” in which the A site has no mRNA that can interact with a new charged tRNA. The non-stop complex cannot be recycled by the normal termination factors.

Instead, the ribosome is rescued by a process called trans-translation. In virtually all bacteria, the rescue system consists of an RNA called transfer-messenger RNA (tmRNA) and a very small protein, small protein B (SmpB). These bind to the stalled complex in such a way that the tmRNA is positioned in the empty A site so that the ribosome can continue translation until it encounters a stop codon embedded in the tmRNA. The ribosome is then recycled, and both the defective mRNA and the polypeptide translated from it are degraded. Similar systems exist in eukaryotes.

CONCLUSION

• The synthesis of polymeric biomolecules can be considered in terms of initiation, elongation, and termination stages. These fundamental processes are typically bracketed by two additional stages: activation of precursors before synthesis and postsynthetic processing of the completed polymer. Protein synthesis follows the same pattern. The activation of amino acids before their incorporation into polypeptides and the posttranslational processing of the completed polypeptide play particularly important roles in ensuring both the fidelity of synthesis and the proper function of the protein product.
• Protein synthesis occurs on the ribosomes, which consist of protein and rRNA. Bacteria have 70S ribosomes, with a large (50S) and a small (30S) subunit. Eukaryotic ribosomes are significantly larger (80S) and contain more proteins.
•  Transfer RNAs have 73 to 93 nucleotide residues, some of which have modified bases. Each tRNA has an amino acid arm with the terminal sequence CCA(3′) to which an amino acid is esterified, an anticodon arm, a TψC arm, and a D arm; some tRNAs have a fifth arm. The anticodon is responsible for the specificity of interaction between the aminoacyl-tRNA and the complementary mRNA codon.
•  The growth of polypeptides on ribosomes begins with the amino-terminal amino acid and proceeds by successive additions of new residues to the carboxyl-terminal end.
• Protein synthesis occurs in four stages.

1. Amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPi . The fidelity of protein synthesis depends on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites.
2. In bacteria, the initiating aminoacyl-tRNA in all proteins is Nformylmethionyl-tRNAfMet . Initiation of protein synthesis involves formation of a complex between the 30S ribosomal subunit, mRNA, GTP, fMet-tRNAfMet , three initiation factors, and the 50S subunit; GTP is hydrolyzed to GDP and Pi .
3. In the elongation steps, GTP and elongation factors are required for binding the incoming aminoacyl-tRNA to the A site on the ribosome. In the first peptidyl transfer reaction, the fMet residue is transferred to the amino group of the incoming aminoacyl-tRNA. Movement of the ribosome along the mRNA then translocates the dipeptidyl-tRNA from the A site to the P site, a process requiring hydrolysis of GTP. Deacylated tRNAs dissociate from the ribosomal E site.
4.  4. After many such elongation cycles, synthesis of the polypeptide is terminated with the aid of release factors. At least four high-energy phosphate equivalents (from ATP and GTP) are required to generate each peptide bond, an energy investment required to guarantee fidelity of translation.

REFERENCES

• Lehninger  principles of biochemistry seventh edition By  David L. Nelson and Michael M. Cox
• voets and voets biochemistry 4^th edition
• Life sciences  fundamental and practices sixth edition, pathfinder publication By Pranav Kumar and Usha Mina
• Essential cell biology (fourth edition) by ALBERTS, BRAY, HOPKIN, JOHNSON, LEWIS, RAFF, ROBERTS, WALTER