RNA synthesis, Genetic code, protein synthesis/ Translation, Protein synthesis inhibitor

 

 

                           TRANSCRIPTION /RNA SYNTHESIS

Transcription is a process of making RNA strand from a DNA template,(single strand nucleic acid) and the RNA molecule that is made and called transcript.

                                                                  OR

     RNA is synthesized with the help of DNA in a process called transcription. 

GENE- these are functional unit of DNA that can be translated.

The genetic information stored in DNA and this expressed through RNA.

There are one of two strands of DNA serves as Template, non-coding strand are produces working copies of RNA

Those strand are not participate in transcription which called non-templets coding strand.

Transcription is selective- Entire molecule of DNA is not expressed in transcription, where RNA is synthesised only for some selected regions of DNA.

Ø  The product formed in transcription = Primary transcript (inactive)

Ø  where post transcriptional modifications produces; Splicing (joined two pieces form one long piece),terminal addition( addition of elements in serial), base modification(alters the bases property and function by controlling sequences).

Ø  Produces functionally active RNA molecule.

Enzyme for transcription: there are single enzyme “RNA Polymerase” which are synthesizes all RNA those are present in prokaryotes.

§     The RNA polymerase is a complex holoenzyme with 5 polypeptide subunits- 2 α, 1 β, 1β’ and one sigma factor.

The enzyme without sigma factor is called core enzyme ( α2 ββ’)

The nuclei of eukaryotic cells passes 3 distinct RNA polymerase-

1.     RNA polymerase : they synthesize of large ribosomal RNAs.

2.     RNA polymerase: they senthesize of mRNAs and small nuclear RNAs

3.     RNA   polymerase: they participate in formation of tRNAs and small ribosomal RNAs.

Transcription process- there are 5 process but major 3 stages

1. Promoter binding

2. DNA unwinding

 3. RNA chain initiation

 4. RNA chain elongation

 5. RNA chain termination

Stages of termination-

The process of DNA transcription can be split into 3 main stages: initiation, elongation & termination. These steps are also involved in DNA replication.

Promotor binding- DNA promoter region is a stretch of about 40bp (base pair) adjacent and its including in the transcription as starting point. Promoter have starting point with six- nucleotide-10 sequence, six- nucleotide -35 sequence located approximately 10 nucleotide and 35 nucleotides upstream from start point.

RNA polymerase binds at promoter by sigma subunit.

                                              OR

Transcription is catalysed by the enzyme RNA polymerase, which attaches to and moves along the DNA molecule until it recognises a promoter sequence. This area of DNA indicates the starting point of transcription, and there may be multiple promoter sequences within a DNA molecule. Transcription factors are proteins that control the rate of transcription; they too bind to the promoter sequences with RNA polymerase.

Once bound to the promoter sequence, RNA polymerase unwinds a portion of the DNA double helix, exposing the bases on each of the two DNA strands,.

DNA Unwinding-  A DNA unwinding element (DUE or DNAUE) is the initiation site for the opening of the double helix structure of the DNA at the origin of replication for DNA synthesis

Initiation  :-

Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region (DNA) of a gene called the promoter regeons.

 The signals of the DNA unwind so the enzyme can ‘‘read’’ the bases in one of the DNA strands.

The enzyme is now ready to make a strand of mRNA with a complementary sequence of bases.

Elongation ;-

Elongation is the addition of nucleotides to the mRNA strand.

RNA polymerase reads the unwound DNA strand and builds the new mRNA molecule, using complementary base pairs.

There is a brief time during this process when the newly formed RNA is bound to the unwound DNA. During this process, an adenine (A) in the DNA binds to an uracil (U) in the RNA.

One DNA strand (template strand) is read in a 3′ to 5′ (three-prime to five-prime) direction, provides the template for the new mRNA molecule. Bases can only be added to the 3′ end, so the strand elongates in a 5’ to 3’ direction.

Termination :-

Termination is the ending of transcription, and occurs when RNA polymerase crosses a stop (termination) sequence in the gene. The mRNA strand is complete, and it detaches from DNA. At this point, transcription stops, and the RNA polymerase releases the DNA template.

GENETIC CODE-

·       The genetic code was explained by an American biochemist of Indian Origin Marshall Nirenberg along with W. Holley and HarGobind Khorana they

·       explained properly about genetic code- “For interpretation of genetic code and its function in protein synthesis” and they got nobel  prize.

·       Genetic code is the system of three nucleotide base sequences in mRNA that determines the sequence of amino acid in protein or they act as a code word for amino acids in protein.

·       As DNA is a genetic material, it carries genetic information from cell to cell and from generation to generation.

·       Only four bases (A,T,G,C) in DNA and twenty amino acids in protein, so some combinations of the bases is needed to specify a particular amino acid.

·       The set of such combinations is called genetic code. A base sequence corresponding to a particular amino acid is a codon.

Codon: Codons are a group of three adjacent bases that specify the amino acids of protein.

Properties of Genetic Code

1. The genetic code is a triplet code:

·       A triplet or three-letter code was first suggested by the physicist Gamow in 1954_ According to the triplet code three letters or bases specify one amino acid.

·       Only 4 of the 20 types of amino ssssssacids would be coded by a singlet code. In a two-letter or doublet code two bases would specify one amino acid. Here 16 (4 x 4) of the 20 amino acids can be specified, but there would be unclear determination of a number of amino acids.

·       Thus 64 (4 x 4 x 4) distinct triplets of purine and/or pyrimidine bases determine the 20 amino acids.

·       Experimental evidence shows that the code is a triplet one, and that 61 of the 64 codons code for individual amino acids during protein synthesis.

2. The code is non-overlapping:

·       The DNA molecule is a long chain of nucleotides it could be read either in an overlapping or non-overlapping manner. In the non-over- lapping code six nucleotides would code for two amino acids, while in the overlapping code up to four could be coded.

·       In the non-overlapping code each letter is read only once while in the over- lapping code it would be read three times, each time as a part of a different word. Mutational changes in one letter would affect only one word-in the non- overlapping code while it would affect three words in the overlapping code.

1.     The code is comma less:

·       A code with commas could be represented as follows (the X represents a base acting as a comma). DDD X CUC X GUA X UCC X ACC------Bases

               Phe Leu Val Ser Thr -----Amino acids (phenylalanine, leucine, valine, serine, threonine)

·       A mutation resulting in an addition or deletion of a base would affect only one amino acid of the polypeptide chain. The total genetic message would be only slightly changed.

DUU X –UC X GUA X UCC X ACC ---Bases

Phe   Changed  Val  Ser  Thr ----amino Acids

·       In such a code any mutation involving a deletion of a base (-C) would result in a drastic change in the genetic message.

UUU UCG UAU CCA CC ----Bases

 Phe Ser Tyr Pro ----Amino acids

The entire series of amino acids following the deletion would change.

2.     The code is non ambiguous (unclear):

A particular codon will always code for the same amino acid. In an ambiguous code, the same codon could coded two or more than one codon (i.e. the code is degenerate), the same codon shall not code for 2 or more different amino acids (non-ambiguous) except when same codon in the nucleus and mitochondria may code for different amino acids.

5. The code is universal:

·       The genetic code is valid for all organisms ranging from bacteria to man. So we said to be universal.

·       Xenopus laevis (amphibian, an aquatic frog) and guinea pig use the same genetic code. (Except mitochondria and ciliate protozoa)

·       In ciliate protozoa (Mycoplasma capricolum-species of bacteria) in this genetic code, codon UAA and UAG specify glutamine instead of stop signals. In future more such cases may be discovered, showing diversity in the genetic code.

6. The code has polarity:

·       If a gene is to specify the same protein repeatedly it is essential that the code must be read between fixed start and end points. These points are the initiation and the termination codons.

·       It is also essential that the code must be read in a fixed direction. It is obvious that if the code is read in opposite directions it would specify two different proteins, since the codons would have reversed base sequences.

·       If the message given below is read from left to right the first codon, UCA (first codon from left 5’ end), would specify serine.

·       If read from right to left the codon would become ACU and would specify threonine. The sequence of amino acids constituting the protein would undergo a drastic change if the code is read in the opposite direction. The available evidence indicates that the message in mRNA is read in the 5'→3' direction.

7. Codons and anticodons:

·       During translation the codons of mRNA pair with complementary anticodons of tRNA. Then mRNA is read in a polar manner in the 5'→3' direction the codons are also written in the 5'→3' direction. Thus the codon AUG is written as 5'AUG3'.

·       Where anticodon on tRNA should therefore be written as 5'CAU3', In such a configuration the first bases of both codon and anticodon would be the ones at the 5' end and third bases at the 3' end.

·       Base number 1 2 3 Codon (mRNA) 5’ A U G 3’ Anticodon (tRNA) 3’ U A C 5’ Base number 3 2 1.

·       The anticodon is written in the 3'→5' direction so as to bring about an easier correlation between the bases of the codon and anticodon.

·       Thus the anticodon for AUG is written as 3' UAC 5' or, more simply, UAC. Here the first letter in the codon is at the 5' end and the first letter of the anticodon at the 3' end.

8. Initiation codons:

·       The starting amino acid in the synthesis of most protein chains is methionine (eukaryotes) or N formyl methionine (prokaryotes).

·       MethionyI or N-formyJ methionyl-tRNA specifically binds to initiation sites containing the AUG codon. This codon is therefore called the initiation codon.

9. Termination codons:

·       Three of the 64 codons do not specify any tRNA and were hence called nonsense codons. These codons are VAG (amber), VAA (ochre) and UGA (opal or umber).

·       Since they bring about termination of polypeptide chain synthesis they are also called termination codons.

TRANSLATION /PROTEIN SYNTHESIS

The process of protein synthesis provides cells with building blocks and regulatory molecules essential for cellular function and survival. ( cell makes protein based on the message contained with in DNA). Where –

·       DNA is only present in nucleus

·       And proteins are only made outside the nucleus in the cytoplasm .

Synthesis:- A molecular cousin of DNA & RNA is used for carry messeges.

Translating the genetic information encoded in mRNA molecules into polypeptide chains is a complex, multistep procedure involving numerous regulatory factors, auxiliary components, and specialized nano machines (ribosomes).

TRANSLATION:-

Definition -Formation of protein from mRNA known as translation/protein synthesis/ polypeptide synthesis.

Requirements for translation process : -

1.     Amino  acids

2.     Ribosomes- They have two subunit example; 60s (big) 40s (small).

3.     m- RNA. They have genetic information in the form of codon.

4.      t- RNA - They transfer amino acid to growing peptide chain, It has anticodons, which is easily recognised the codon of mRNA .

5.     Energy sources - ATP and GTP

6.     Protein factors- They needed for initiation, elongation, and termination.

Steps involve in Protein synthesis

                 I.          Activation of amino acid ( in 2 step)

               II.          Protein synthesis propane – Initiation, Elongaton, Termination

Step 2- Elongation

·       Elongation is done by formation of peptide linkage.

·       t-RNA brings amino acids  to code the pairing of codon and anti-codon and then they join from amino acid and form of protein.

·       So the first t- RNA is attached on P- site, when the AUG codon comes then start the process.

·       t -RNA goes to E- site then they gave their amino acid to A - site.

·       This process is continued and many amino acid join by peptide linkage. 

Step 3: Termination

·       At this step terminating factors comes and join at B-site and terminate everything.

·       Terminating factors –R1, R2, S

Protein synthesis inhibitors-

Protein synthesis, a long process and they includes different enzymes and structural change in organisms.

Some antibacterial classes inhibit bacterial protein synthesis by interfering with the 30s or 50s subunit Antibiotics target three different steps which include initiation, formation of the 70s, and elongation process of making polypeptides. Antibiotics that inhibit or interferes with bacterial protein synthesis example; Aminoglycosides, Macrolides, Tetracycline, Oxazolidinone, and Chloramphenicol.

1.Aminoglycosides:

Aminoglycosides only interact with the nucleic acid bases but not with the phosphate backbone. Electronic and Stearic constraints should be satisfied in case of not binding with RNA. 

2.Tetracycline: They consist of A, B, C, and D nucleus rings in which several functional groups are attached.  They interact with the ribosome and show reversible bacteriostatic properties by inhibiting bacterial protein synthesis. 

3. Chloramphenicol: Chloramphenicol was referred to as chloromycetin (Streptomyces venezuelae). As it broad-spectrum antibiotic it is used to treat both Gram-positive and Gram-negative infections and it is very stable. They are protein synthesis inhibitors that prevent the bacteria from peptide chain elongation.

4. Macrolides: Macrolides that is used in treating Gram-positive infections. Macrolides are protein synthesis inhibitors as it binds to ribosomal subunit in peptidyl transferase center and causing cell lysis by inhibiting protein synthesis.

5. Oxazolidinones: Oxazolidinones are a newer class of antibiotics. Oxazolidinone acts by inhibiting initiation complex, binding to P-site, and translocation of peptidyl-tRNA from A-site to P-site.

 

Mechanism of action of protein synthesis inhibitors-

Protein synthesis inhibitors usually act at the ribosomal level in the translation process of protein synthesis that includes initiation, elongation, and termination.

 In both prokaryotes and eukaryotes, ribosomes are the main site for protein synthesis. Mainly, tRNA binds to three sites of mRNA complex; A-site or aminoacyl site, Peptidyl site or P-site, and E site or Exit site.