Translation , Transcription and Transduction

The formation of mRNA form DNA template under certain proteins as well as enzymes is called Translation.

In translation, messenger RNA (mRNA)—produced by transcription from DNA—is decoded by aribosome to produce a specific amino acid chain, or polypeptide. The polypeptide later folds into anactive protein and performs its functions in the cell. The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome. The entire process is a part of gene expression.

In brief, translation proceeds in four phases:

Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.

Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon.

Translocation:The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain.

Termination: When a stop codon is reached, the ribosome releases the polypeptide.

In bacteria, translation occurs in the cell's cytoplasm, where the large and small subunits of theribosome bind to the mRNA. In eukaryotes, translation occurs in the cytosol or across the membrane of the endoplasmic reticulum in a process called vectorial synthesis. In many instances, the entire ribosome/mRNA complex binds to the outer membrane of the rough endoplasmic reticulum (ER); the newly created polypeptide is stored inside the ER for later vesicle transport and secretion outside of the cell.

Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA, do not undergo translation into proteins.

A number of antibiotics act by inhibiting translation. These include anisomycin, cycloheximide,chloramphenicol, tetracycline, streptomycin, erythromycin, and puromycin. Prokaryotic ribosomes have a different structure from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial infections without any harm to a eukaryotic host's cells.

Basic mechanisms

The basic process of protein production is addition of one amino acid at a time to the end of a protein. This operation is performed by a ribosome. The choice of amino acid type to add is determined by an mRNA molecule. Each amino acid added is matched to a three nucleotide subsequence of the mRNA. For each such triplet possible, the corresponding amino acid is accepted. The successive amino acids added to the chain are matched to successive nucleotide triplets in the mRNA. In this way the sequence of nucleotides in the template mRNA chain determines the sequence of amino acids in the generated amino acid chain.[1] Addition of an amino acid occurs at the C-terminus of the peptide and thus translation is said to be amino-to-carboxyl directed.[2]

The mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to the ribosomes. The ribonucleotides are "read" by translational machinery in a sequence of nucleotide triplets called codons. Each of those triplets codes for a specific amino acid.

The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a multisubunit structure containing rRNA and proteins. It is the "factory" where amino acids are assembled into proteins. tRNAs are small noncoding RNA chains (74-93 nucleotides) that transport amino acids to the ribosome. tRNAs have a site for amino acid attachment, and a site called an anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes for their cargo amino acid.

Aminoacyl tRNA synthetases (enzymes) catalyze the bonding between specific tRNAs and the amino acids that their anticodon sequences call for. The product of this reaction is an aminoacyl-tRNA. This aminoacyl-tRNA is carried to the ribosome by EF-Tu, where mRNA codons are matched through complementary base pairing to specific tRNAanticodons. Aminoacyl-tRNA synthetases that mispair tRNAs with the wrong amino acids can produce mischarged aminoacyl-tRNAs, which can result in inappropriate amino acids at the respective position in protein. This "mistranslation"[3] of the genetic code naturally occurs at low levels in most organisms, but certain cellular environments cause an increase in permissive mRNA decoding, sometimes to the benefit of the cell.

The ribosome has three sites for tRNA to bind. They are the aminoacyl site (abbreviated A), the peptidyl site (abbreviated P) and the exit site (abbreviated E). With respect to the mRNA, the three sites are oriented 5’ to 3’ E-P-A, because ribosomes move toward the 3' end of mRNA. The A site binds the incoming tRNA with the complementary codon on the mRNA. The P site holds the tRNA with the growing polypeptide chain. The E site holds the tRNA without its amino acid. When an aminoacyl-tRNA initially binds to its corresponding codon on the mRNA, it is in the A site. Then, a peptide bond forms between the amino acid of the tRNA in the A site and the amino acid of the charged tRNA in the P site. The growing polypeptide chain is transferred to the tRNA in the A site. Translocation occurs, moving the tRNA in the P site, now without an amino acid, to the E site; the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P site. The tRNA in the E site leaves and another aminoacyl-tRNA enters the A site to repeat the process.

After the new amino acid is added to the chain, and after the mRNA is released out of the nucleus and into the ribosome's core, the energy provided by the hydrolysis of a GTP bound to the translocase EF-G (in prokaryotes) and eEF-2 (in eukaryotes) moves the ribosome down one codon towards the 3' end. The energy required for translation of proteins is significant. For a protein containing n amino acids, the number of high-energy phosphate bonds required to translate it is 4n-1[citation needed]. The rate of translation varies; it is significantly higher in prokaryotic cells (up to 17-21 amino acid residues per second) than in eukaryotic cells (up to 6-9 amino acid residues per second).

In activation, the correct amino acid is covalently bonded to the correct transfer RNA (tRNA). The amino acid is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, it is termed "charged". Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with the help of initiation factors (IF). Termination of the polypeptide happens when the A site of the ribosome faces a stop codon (UAA, UAG, or UGA). No tRNA can recognize or bind to this codon. Instead, the stop codon induces the binding of a release factor protein that prompts the disassembly of the entire ribosome/mRNA complex.

Genetic code

AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG

Starts = ---M---------------M---------------M----------------------------

Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG

Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG

Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG

TranscriptionTranscription is the first step of gene expression, in which a particular segment of DNA is copied into RNA (mRNA) by the enzyme RNA polymerase.

Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementarylanguage. The two can be converted back and forth from DNA to RNA by the action of the correct enzymes. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript.

Transcription proceeds in the following general steps:

One or more sigma factor protein binds to the RNA polymerase holoenzyme, allowing it to bind to promoter DNA.

RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides.

RNA polymerase adds matching RNA nucleotides to the complementary nucleotides of one DNA strand.

RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.

Hydrogen bonds of the untwisted RNA-DNA helix break, freeing the newly synthesized RNA strand.

If the cell has a nucleus, the RNA may be further processed. This may includepolyadenylation, capping, and splicing.

The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear porecomplex.

The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene transcribed encodes a protein,messenger RNA (mRNA) will be transcribed; the mRNA will in turn serve as a template for the protein's synthesis through translation. Alternatively, the transcribed gene may encode for either non-coding RNA (such as microRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), or other enzymatic RNA molecules calledribozymes.[1] Overall, RNA helps synthesize, regulate, and process proteins; it therefore plays a fundamental role in performing functions within a cell.

In virology, the term may also be used when referring to mRNA synthesis from an RNA molecule (i.e., RNA replication). For instance, the genome of a negative-sense single-stranded RNA (ssRNA -) virus may be template for a positive-sense single-stranded RNA (ssRNA +). This is because the positive-sense strand contains the information needed to translate the viral proteins for viral replication afterwards. This process is catalysed by a viral RNA replicase.

Reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is reverse transcribed into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase.

In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. The enzyme ribonuclease H then digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure ("cDNA"). The cDNA is integrated into the host cell's genome by the enzyme integrase, which causes the host cell to generate viral proteins that reassemble into new viral particles. In HIV, subsequent to this, the host cell undergoes programmed cell death, or apoptosis of T cells.[21] However, in other retroviruses, the host cell remains intact as the virus buds out of the cell.

Transduction:

Definition - Unlike transformation in which the naked DNA is transferred in transduction DNA is carried by a bacteriophage. OR

In transduction, DNA is transferred from cell to cell through the agency of viruses

NOTE :- All phages can be transducer and not all bacteria are transducible

EXAMPLES OF BACTERIA

Transduction has been found to occur in a variety of prokaryotes, including certain species of the Bacteria: Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus, Rhodobacter, Salmonella, Staphylococcus, and Xanthobacter, as well as M ethanobacterium thermoautotrophicum.

Mechanism of Transduction

Bacteriphage

Firstly Descovered in 1915 by Fredrick Twort and two years later by Felix d’Herelle.

Means bacteria eater.

A virus that infects certain type of bacteria and replicates within them

Types of bacterio-phage

Virulent: capable of causing infection and eventually destruction and death of the bacterial cell. These follow the lytic cycle. e.g. T4 host E.coli.

Temperate: does not cause destruptic infection instead phage DNA is incorporated into bacterium DNA and is replicated with it and after some cycle become virulent cause lysis.

e.g. lambda phage.

Life cycle of bacteriophage

There are two types of transduction:

Generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle

1. A lytic bacteriophage adsorbs to a susceptible bacterium.

2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes

3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid instead of a phage genome by mistake.

4. The bacteriophages are released.

5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .

• It transfers genetic material from one bacterial cell to another and alter the genetic characteristics.

• For example: In specialised transduction the gal gene, a cell lacking ability to metabolize galactose could aquire the ability .

• It shows the evolutionary relationship between the prophage and host bacterial cell.

• Prophage can exist in a cell for a long period suggests a similar possible mechanism for the viral origin of cancer.

• It provides a way to study the gene linkage.