9 Mechanism of Protein Synthesis

9 MECHANISM OF PROTEIN SYNTHESIS  The process of protein synthesis consists of two major steps: (A) Transcription or synthesis of mRNA on DNA (B) Translation or synthesis of proteins along mRNA (A) Transcription  The transfer of genetic information from DNA to mRNA is known as transcription.  The segment of DNA that takes part In transcription is called transcription unit. It has three components. (i) A Promoter; (ii)The Structural gene & (iii)A Terminator Promoter sequences are present upstream (5' end) of the structural genes of a transcription unit.  The binding sites for RNA polymerase lie within the promoter sequence.  Certain short sequences within the promoter sites are conserved. In prokaryotes, 10 bp upstream from, the start point lies a conserved sequence described as –10 sequence TATAAT or "Pribnow box" and –35 sequence TTGACA as "Recognition sequence". Fig: Prokaryotic promoter sites for transcription Fig: The prokaryotic σ factor is required for accurate initiation of transcription. Fig: Schematic structure of a transcription unit  Structural gene is part of that DNA strand which has 3'  5' polarity, as transcription occur in 5'  3' direction. This strand of DNA that direct the synthesis of mRNA is called template strand. The complementary strand is called non-template or coding strand, it is identical in base sequence to RNA transcribed from the gene, only with U in place of T.  Terminator is present at 3' end of coding strand and defines the end of the process of transcription. Mechanism of transcription  RNA polymerase binds to promoter region of the DNA and the process of transcription begins.  RNA polymerase moves along DNA helix and unwinds it.  One of the two strands of DNA serves as a template for RNA synthesis.  This results in the formation of complementary RNA strand.  It is formed at a rate of about 40 to 50 nucleotides per second.  RNA synthesis comes to a stop when RNA polymerase reaches the terminator sequence.  The transcription enzyme, i.e., RNA polymerase is only of one type in prokaryotes and can transcribe all types of RNAs.  RNA polymerase is a holoenzyme that is represented as (2').  Molecular weight of holoenzyme is 4,50,000.  The enzyme without subunit is referred to as core enzyme.  Though, the core enzyme is capable of transcribing DNA into RNA but transcription  starts non specifically at any base on DNA. It is subunit which confers specificity. Rho factor ( ) is required for termination of transcription. (a) Sigma Factor : Binds to the promoter site of DNA and it initiates transcription. (b) Core Complex : It continues the transcription. (c) Rho Factor : It terminates transcription, its molecular weight is 55,000. Transcription in Eukaryotes  In eukaryotes, the promotor site is recognised by presence of specific nucleotide sequence called TATA box or Hogness box (7 base pair long -TATATAT or TATAAAT) located 20 bp upstream to the start point. Another sequence is CAAT box present between -70 and -80 bp.   In eukaryotes, the RNA polymerases are of three types, i.e., RNA polymerase I, RNA polymerase II and RNA polymerase III. Functions of different RNA polymerases in eukaryotes are given below: (i) RNA polymerase-I  5.8 S, 18 S, 28 S rRNA synthesis (ii) RNA polymerase-II  Hn RNA (heterogenous nuclear RNA), mRNA synthesis (iii) RNA polymerase-III  tRNA, Sc RNA (small cytoplasmic RNA), 5S rRNA and Sn RNA (small nuclear RNA) synthesis.  The gene in eukaryotes, however, is made of several pieces of base sequences coding for amino acids called exonic DNA, separated by stretches of non-coding sequences, commonly called intronic DNA. Thus, the information on the eukaryotic gene for assembling a protein is not continuous but split.  This discovery is the result of works of Richard J. Roberts and Philip Sharp. FIG: DNA Is Transcribed to Form RNA DNA is partially unwound by RNA polymerase so that one strand can serve as a template for RNA synthesis. The RNA transcript is formed and then peels away, allowing the DNA that has already been transcribed to rewind into a double helix.  They shared 1993 Nobel Prize for Physiology and Medicine for the discovery of 'split genes' in higher organisms.  Most eukaryotic genes contain very long base sequences, all of which do not necessarily form mature mRNA.  The coding DNA sequences of the gene are called exons and the intervening noncoding DNA sequences are called introns.  All introns have GU at 5' end and AG at 3' end. Depending on the size of gene, the number and length of exons may vary from a few to more than fifty, alternating with stretches of DNA that contain no genetic information introns.  The nascent RNA synthesized by RNA polymerase-II is called hnRNA (heterogenous nuclear RNA).  It contains both unwanted base sequences (transcribed from introns) alternated with useful base sequences (transcribed from exons).  This primary transcript is converted into functional mRNA after post-transcriptional 1. processing which involves 3 steps: Modification of 5' end by Capping: Capping at 5' end occurs rapidly after the start of transcription.  The guanosine methylated at 7th position is added at 5' with the help of enzyme guanyl transferase. Cap is essential for formation of mRNA-ribosome complex.  Translation is not possible if cap is lacking, because cap is identified by 18 S 2. 3. rRNA of ribosome unit. Polyadenylation at 3' end (Tailing) : Poly (A) is added to 3' end of newly formed hn RNA with the help of enzyme Poly A polymerase. It adds about 200-300 adenyl ate residues. Splicing of hnRNA (Tailoring) : In eukaryotes the coding sequences of RNA (exons) are interrupted by non coding sequences (introns). Small nuclear RNA (snRNA) + Protein complex called small nuclear ribonucleo protein or SnRNPs (or snurps) play important role in this process. Here the introns are removed and exons come in one plane. This process is called splicing through which a mature mRNA is produced.  Normally, mRNA carries the codons of single complete protein molecule (monocistronic mRNA) in eukaryotes, but in prokaryotes, it carries codons from several adjacent DNA cistrons and becomes much longer in size (polycistronic mRNA). Fig: The Spliceosome: An RNA Splicing Machine Small nuclear ribonucleoprotein particles (snRNPs) bind to consensus sequences bordering the introns on pre-mRNA transcripts. Other proteins then bind, forming a large complex called a spliceosome. This structure determines the exact position of each cut in the pre-mRNA with great precision. Fig: Post-transcriptional processing in eukaryotes Genetic Code  Term was coined by Gamow.     DNA (or RNA) carries all the genetic information. It is expressed in the form of proteins. Proteins are made up of 20 different types of essential amino acids. The information about the number and sequence of these amino acids forming protein    is present in DNA and is passed on to mRNA during transcription. Genetic code is a mRNA sequence containing coded information for one amino acid and consists of three nucleotides (triplet). Thus, for twenty amino acids, 64 (4 × 4 × 4 or 43 = 64) minimum possible permutations are available. This important discovery was the result of experiments by Marshall W. Nirenberg and J. Heinrich Matthaei and later by H.G. Khorana. Nirenberg and Khorana shared the 1968 Nobel Prize with R.W. Holley who gave details of tRNA structure. Nirenberg and Mathaei used a synthetic poly (U)RNA and deciphered the code by translating this as polyphenylalanine. Hargobind Khorana, using synthetic DNA, prepared polynucleotide with known repeating sequence, e.g., CUCUCUCUCUCU, it produced only two amino acids, leucine (CUC) and serine (UCU). Properties of Genetic Code 1. Triplet code. Three adjacent nitrogen bases constitute a codon which specifies the placement of one amino acid in a polypeptide. 2. Start signal. Polypeptide synthesis is signalled by two initiation codons-AUG or rarely GUG. The first or initiating amino acid is methionine. The initiating codon on mRNA for methionine is AUG. Initiating methionine occurs in formylated state in prokaryotes. At other positions methionine is non-formylated. Both these methionines are carried by different tRNAs. Rarely, GUG also serves as initiation codon. It normally codes for valine but if present at initiating position, it would code for methionine. So, GUG is an 3. ambiguous codon . Stop signal. Polypeptide chain termination is signalled by three termination codonsUAA (ochre), UAG (amber) and UGA (opal). They do not specify any amino acid and were hence called as nonsense codons. Whenever present in mRNA, these bring about termination of polypeptide chain and thus, act as stop signals. Codon UAA, UAG, UGA, AUG, and GUG are also called as punctuation codons. 4. Commaless. The genetic code is continuous and does not possess pauses (meaningless base) after each triplet. If a nucleotide is deleted or added, the whole genetic code will read differently. Thus, a polypeptide having 50 amino acids shall be specified by a linear sequence of 150 nucleotides. If a nucleotide is added or deleted in the middle of this sequence, the amino acids before this will be same but subsequent amino acids will be quite different. 5. Universal code. The genetic code is universal – almost every living organism uses the same code (there are a few rare and minor exceptions)  As the same codons code for the same amino acids in all living things, genetic information is transferrable between species. The ability to transfer genes between species has been utilised to produce human insulin in bacteria (for mass production)  The gene responsible for insulin production is extracted from a human cell  It is spliced into a plasmid vector (for autonomous replication and expression) before being inserted into a bacterial cell  The transgenic bacteria (typically E. coli) are then selected and cultured in a fermentation tank (to increase bacterial numbers)  The bacteria now produce human insulin, which is harvested, purified and packaged for human use (i.e. by diabetics) Fig: Insulin Production via Recombinant Gene Transfer 6. 7. 8. Non-overlapping code. Every codon is independent and one codon does not overlap the next codon. Degeneracy of code. Since there are 64 triplet codons and only 20 amino acids, the incorporation of some amino acids must be influenced by more than one codon. Only tryptophan and methionine are specified by single codons. All other amino acids are specified by 2-6 codons. The latter are called degenerate codons. Deciphering the Genetic Code Nirenberg and Matthaei used a test tube protein synthesis system to determine the amino acids specified by synthetic mRNAs of known compositions. Fig: Deciphering the Genetic Code Wobble Hypothesis  A change in nitrogen base at the third position of a codon does not normally cause any change in the expression of the codon because the codon is mostly read by the first two      nitrogen bases (wobble hypothesis of Crick). The mutation that does not cause any change in the expression of the gene is called silent mutation. The position of the third nitrogen base in a codon which does not influence the reading of the codon is termed as Wobble position. It pairs with 1st position in anticodon. It means specificity of codon is determined by first two bases. Wobbling helps one tRNA to read more than one codon and thus, provides economy in number of tRNA molecules at the time of translation. The insertion of single base G between the 3rd and 4th bases produces completely different protein from earlier one.  Similarly, the deletion of single base C at 4th place produces a new chain of amino acids and hence a different protein. MUTATIONS AND GENETIC CODE :  In a hypothetical mRNA, for example, the codons would normally be translated as follows :  Insertion or deletion of one or two nitrogenous bases changes the reading frame from the point of insertion or deletion. Such mutations are called frame shift mutations. However, insertion or deletion of three or its multiple bases insert or delete one or multiple codons hence one or multiple amino acids and reading frame remains unaltered from that point onwards.  This form genetic basis of proof that codon is a triplet and it is read in a contiguous manner. Fig: Assignment of mRNA codons to Amino Acids (B) Translation  It is the mechanism by which the triplet base sequences of m-RNA molecules are converted into a specific sequence of amino acids in a polypeptide-chain. It occurs on ribosomes. (a) Activation of amino acids. In the presence of enzyme aminoacyl -tRNA synthetase (E), specific amino acids (AA) bind with ATP Charging of t-RNA: The AA1-AMP-E1 complex formed in first step reacts with a specific tRNA. Thus, amino acid is transferred to tRNA. As a result, the enzyme and AMP are liberate (b) (i) Formation of polypeptide chain. It is completed in three steps. Chain Initiation. It requires 3 initiation factors in prokaryotes i.e., IF3, IF2, IF1. 9 initiation factors are required in eukaryotes i.e., eIF2, eIF3, eIF1, eIF4A, eIF4B, eIF4C, eIF4D, eIF5, eIF6. (a) Binding of mRNA with smaller subunit of ribosomes (30S/40S). IF3 is involved in prokaryotes, while eIF2 is involved in eukaryotes. It involves interaction between Shine Delgarno sequence and 3' end of 16 S rRNA in prokaryotes. (b) Binding of 30S / 40S-mRNA complex with t-RNA. Non-formylated methionine is attached with tRNA in eukaryotes and formylated methionine in prokaryotes. IF2 is involved in prokaryotes and eIF3 in eukaryotes. GTP (c) Attachment of larger subunit of ribosomes. Initiation factors in eukaryotes -eIFl, eIF4. Initiation factors in prokaryotes -IF1. Fig: The Initiation of Translation Translation begins with the formation of an initiation complex. (ii) Chain elongation.  Ribosomes have two sites for binding amino acyl tRNA (i) Amino acyl or A site (acceptor site) (ii) Peptidyl site or P site (donor site). A second charged tRNA molecule along with its appropriate amino acid approaches the ribosome at A site close to the P site. Its anticodon binds to complementary codon of mRNA chain.     A peptide bond is formed between COOH group of first amino acid (methionine) and NH2 group of second amino acid. The formation of peptide bond requires energy and is catalysed by enzyme peptidyl transferase. The initiating formyl methionine or methionine tRNA can bind only with P site. All other newly coming aminoacyl tRNA bind to A site.  The elongation factor required for prokaryotes are EF-Tu and EF-Ts and in eukaryotes eEF1. Translocation is movement of ribosome on mRNA. It requires EF -G in prokaryotes and eEF2 in eukaryotes Fig: The Elongation of Translation The polypeptide chain elongates as the mRNA is translated. (iii) Chain termination. The termination of polypeptide is signalled by one of the three terminal codons in the mRNA. The three terminal codons are UAG (Amber), UAA (Ochre) and UGA (Opal).   A GTP dependent factor known as release factor is associated with termination codon. It is eRF1 in eukaryotes and RF1 and RF2 in prokaryotes. At the time of termination, the terminal codon immediately follows the last amino acid codon. After this, the polypeptide chain, tRNA, mRNA are released and the subunits of ribosomes get dissociated. Fig: The Termination of Translation Translation terminates when the A site of the ribosome encounters a stop codon on the mRNA. Fig: Translation in bacteria begins with the formation of the initiation complex, which includes the small ribosomal subunit, the mRNA, the initiator tRNA carrying N-formyl-methionine, and initiation factors. Then the 50S subunit binds, forming an intact ribosome. Fig: (a) In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for a rapid cellular response to an environmental cue. (b) In eukaryotes, transcription is localized to the nucleus and translation is localized to the cytoplasm, separating these processes and necessitating RNA processing for stability. Fig: Destinations for Newly Translated Polypeptides in a Eukaryotic Cell Signal sequences on newly synthesized polypeptides bind to specific receptor proteins on the outer membranes of the organelles to which they are directed. Once the protein has bound to it, the protein enters the organelle through a channel in the membrane.  Gene expression is the mechanism at the molecular level by which a gene is able to express itself in the phenotype of an organism.  In the translation unit, mRNA has some sequences that are not translated and are referred to as untranslated regions (UTR).  The UTRs are present at both 5' end (before start codon) and at 3' end (after stop codon).  They are required for efficient translation process Fig: Posttranslational Modifications of Proteins Most polypeptides must be modified after translation in order to become functional proteins.