TRANSCRIPTION

 TRANSCRIPTION IN PROKARYOTES

• Transcription is the process by which the information in a strand of DNA is copied into a new molecule of messenger RNA (mRNA).
• During replication entire genome is copied but in transcription only the selected portion of genome is copied.

ENZYME INVOLVED IN TRANSCRIPTION

• RNA is synthesized by a single RNA polymerase enzyme which contains multiple polypeptide subunits.
• In E. coli, the RNA polymerase has subunits: two α, one β, one β’ and one ω and σ subunit (α2ββ’ωσ). This complete enzyme is called as the holoenzyme.
• The σ subunit may dissociate from the other subunits to leave a form known as the core enzyme.

STEPS INVOLVED IN PROKARYOTIC TRANSCRIPTION

Transcription is an enzymatic process. the mechanism of transcription completes in three major steps

1. Initiation:

• closed complex formation
• Open complex formation
• Tertiary complex formation

2. Elongation

3. Termination:

INITIATION:

• The transcription is initiated by RNA polymerase holoenzyme from a specific point called promoter sequence.
• Bacterial RNA polymerase is the principle enzyme involved in transcription.

• The core enzyme bind to specific sequence on template DNA strand called promotor. The binding of core polymerase to promotor is facilitates and specified by sigma (σ) factor. (σ70 in case of E. coli).
• The core polymerase along with σ-factor is called Holo-enzyme ie. RNA polymerase  holoenzyme.
• In case of e. coli, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site.The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes.The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.

i. closed complex:

• Binding of RNA polymerase holoenzyme to the promotor sequence form closed complex

ii. Open Complex: 

• After formation of closed complex, the RNA polymerase holoenzyme separates 10-14 bases extending from -11 to +3 called melting. So that open complex is formed. This changing from closed complex to open complex is called isomerization.

iii. Closed Complex: 

• RNA polymerase starts synthesizing nucleotide. It does not require the help of primase.
• If the enzyme synthesize short RNA molecules of less than 10 bp, it does not further elongates which is called abortive initiation. This is because σ 3.2 acting as mimic of RNA and it lies at middle of RNA exit channel in open complex.
•   When the RNA polymerase manage to synthesize RNA more than 10 bp long, it eject the σ 3.2 region and RNA further elongates and exit from RNA exit channel. This is the formation of tertiary complex.

ELONGATION

The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core RNA polymerase enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. Since the base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components, RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely. The synthesized RNA is proof reads by Hydrolytic editing. For this the polymerase back track by one or more nucleotide and cleave the RNA removing the error and synthesize the correct one. The Gre factor enhance this proof reading process.

TERMINATION

There are two mechanism of termination.

i) Rho - independent:

• In this mechanism, transcription is terminated due to specific sequence in terminator DNA.
• The terminator DNA contains invert repeat which cause complimentary pairing as transcript RNA form hair pin structure.
• This invert repeat is followed by larger number of TTTTTTTT(~8 bp) on template DNA. The uracil appear in RNA. The load of hair pin structure is not tolerated by A=U base pair so the RNA get separated from RNA-DNA heteroduplex.

Rho - Dependent termination: 

• In this mechanism, transcription is terminated by rho (ρ) protein.
• It is ring shaped single strand binding ATpase protein.
• The rho protein bind the single stranded RNA as it exit from polymerase enzyme complex  and hydrolyse the RNA from enzyme complex.
• The rho protein does not bind to those RNA whose protein is being translated. Rather it bind to RNA after translation.
• In bacteria transcription and translation occur simultaneously so the rho protein bind the RNA after translation has completed but transcription is still ON.       

 TRANSCRIPTION IN EUKARYOTES

• Transcription is the process by which the information in a strand of DNA is copied into a new molecule of RNA.
• It is the first step of gene expression, in which a particular segment of DNA is copied into RNA (especially 
• 'RNA) by the enzyme RNA polymerase.

ENZYME INVOLVED IN THE EUKARYOTIC TRANSCRIPTION

• RNA polymerase I (RNA Pol I) is located in the nucleolus and transcribes the 28S, 18S, and 5.8S rRNA genes.
• RNA polymerase II (RNA Pol II) is located in the nucleoplasm and transcribes protein-coding genes, to yield pre-mRNA, and also the genes encoding small nucleolar RNAs (snoRNAs) involved in rRNA processing and small nuclear RNAs (snRNAs) involved in mRNA processing, except for U6 snRNA.
• RNA polymerase III (RNA Pol III) is also located in the nucleoplasm. It transcribes the genes for tRNA, 5S rRNA, U6 snRNA, and the 7S RNA associated with the signal recognition particle (SRP) involved in the translocation of proteins across the endoplasmic reticulum membrane.
• Each of the three eukaryotic RNA polymerases contains 12 or more subunits and so these are large complex enzymes.
• The genes encoding some of the subunits of each eukaryotic enzyme show DNA sequence similarities to genes encoding subunits of the core enzyme of E. coli RNA polymerase.
• However, four to seven other subunits of each eukaryotic RNA polymerase are unique in that they show no similarity either with bacterial RNA polymerase subunits or with the subunits of other eukaryotic RNA polymerases.

PROCESS OF EUKARYOTIC TRANSCRIPTION

The basic mechanism of RNA synthesis by these eukaryotic RNA polymerases can be divided into the following phases:

a) INITIATION PHASE: 

• PAPER 

b) ELONGATION PHASE:

TFIIH has two functions:

1. It is a helicase, which means that it can use ATP to unwind the DNA helix, allowing transcription to begin.
2. In addition, it phosphorylates RNA polymerase II which causes this enzyme to change its conformation and dissociate from other proteins in the initiation complex.

• The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain (CTD) of the RNA polymerase II molecule.
• Interestingly, only RNA polymerase II that has a non-phosphorylated CTD can initiate transcription but only an RNA polymerase II with a phosphorylated CTD can elongate RNA.
• RNA polymerase II now starts moving along the DNA template, synthesizing RNA, that is, the process enters the elongation phase.
• TF II S is used to increase the speed of the transcription process. 
• RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on an incoming ribonucleoside 5-triphosphate.
• The RNA molecule made from a protein-coding gene by RNA polymerase II is called a primary transcript.

TERMINATION:

(PAPER)

RESTRICTION ENZYMES

RESTRICTION ENZYMES

A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a random or specific recognition sites known as restriction sites. In bacteria, restriction enzymes form a combined system (restriction + modification system) with modification enzymes that methylate the bacterial DNA. Methylation of bacterial DNA at the recognition sequence typically protects the own DNA of the bacteria from being cleaved by restriction enzyme

There are two different kinds of restriction enzymes:

 (1) Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I, exonuclease II etc. 

(2) Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI etc

EXONUCLEASES AND IT TYPES: 

EXONUCLEASE III: 

• Exonuclease III is a globular enzyme which has 3’→5’ exonuclease activity in a double stranded DNA. 
• The template DNA should be double stranded and the enzyme does not cleave single stranded DNA. 
• The enzyme shows optimal activity with blunt ended sequences or sequences with 5’ overhang. 
• Exonuclease III enzyme has a bound divalent cation which is essential for enzyme activity.
• The mechanism of the enzyme can be affected by variation in temperature, monovalent ion concentration in the reaction buffer, and structure and concentration of 3’termini. 
• The enzyme shows optimal activity at 37°C at pH 8.0.

MUNG BEAN NUCLEASES:

• As the name suggest, this nuclease enzyme is isolated from mung bean sprouts (Vigna radiata). 
• Mung bean nuclease enzymes can degrade single stranded DNA as well RNA. Under high enzyme concentration, they can degrade double stranded DNA, RNA or even DNA/RNA hybrids.
•  Mung bean nuclease can cleave single stranded DNA or RNA to produce 5’-phosphoryl mono and oligonucleotides.
• It requires Zn2+ ion for its activity and shows optimal activity at 37°C. 
• The enzyme works in low salt concentration (25mM ammonium acetate) and acidic pH (pH 5.0). 
• Treatment with EDTA or SDS results in irreversible inactivation of the enzyme. Mung bean nuclease is less robust than S1 nuclease and easier to handle. It has been used to create blunt end DNA by cleaving protruding ends from 5’ ends. This enzyme cannot produce nicks in a double stranded DNA but at higher concentration, it can generate nicks and cleave double stranded DNA.

RNases: 

Ribonucleases are a class of hydrolytic enzymes that catalyzes both the in vivo and in vitro degradation of ribonucleic acid (RNA) molecules into smaller components. Ribonucleases are classified into two types, Exoribonucleases and endoribonucleases. 

Exoribonucleases: The exoribonuclease is an exonuclease ribonuclease that degrades RNA by removing terminal nucleotides from either the 5′ end or the 3′ end of the RNA molecule.

Endoribonucleases: The endonuclease ribonuclease cleaves RNA molecules internally. It can cleave either single-stranded or double-stranded RNA, depending on the enzyme.

RNases I:

RNases A:

RNases H:

T4 PHOSPHO NUCLEOTIDE KINASE:

Polynucleotide kinases (PNK) are one such DNA modification enzymes that add phosphate groups to nucleic acid molecules. They transfer the gamma phosphate group from adenosine triphosphate (ATP) to the 5’ hydroxyl termini of DNA or RNA.

                        ATP + 5'-dephospho-DNA ${\displaystyle \rightleftharpoons }$ ADP + 5'-phospho-DNA

Thus, the two substrates of this enzyme are ATP and 5'-dephospho-DNA, whereas its two products are ADP and 5'-phospho-DNA. Polynucleotide kinase is a T7 bacteriophage (or T4 bacteriophage) enzyme that catalyzes the transfer of a gamma-phosphate from ATP to the free hydroxyl end of the 5' DNA or RNA. The resulting product could be used to end-label DNA or RNA, or in ligation reactions.

DUAL ROLE OF T4 Polynucleotide Kinase

The dual roles—kinase and phosphatase—performed by the enzyme reside in its separate domains. The N-terminal is a 5′-kinase domain with a nucleotide-binding motif GXXXXGK(S/T) and a C-terminal is a 3′-phosphohydrolase domain.

TYPES OF RESTRICTION ENZYMES

Restriction enzymes are traditionally classified into four types on the basis of subunit composition, cleavage position, sequence specificity and cofactor requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level, there are many more than four different types.

TYPE 1

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but this type is not used as like as Type 2. 

Enzyme Activity:

Type I R–M enzymes are pentameric proteins of composition 2R+2M+S. They require ATP, Mg2+ and S-adenosylmethionine (SAM) for activity and display both REase and MTase activities. A trimer of 2M+S acts solely as an MTase