Instrumentation 6

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Microscopy is the study of objects or samples that are too small to be seen by the naked eye. There are several types of microscopy, each with its own advantages and limitations. Here are the main types of microscopy: 1. Optical microscopy: This is the most common type of microscopy, which uses visible light to illuminate a sample. Optical microscopy can be further divided into several subtypes, such as brightfield, darkfield, phase contrast, fluorescence, and confocal microscopy. Optical microscopy is a technique that uses visible light to observe the sample under a microscope. It consists of several components, including an objective lens, an eyepiece lens, and a light source. The working of optical microscopy involves the following steps. The sample to be viewed is prepared by fixing it onto a glass slide and adding a stain or dye to enhance its contrast. The light source, located beneath the sample, emits light that is directed through the condenser lens to focus the light o...
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STD 12th/Chapter 6 DNA Replication, Transcription & Translation. (quick revision)

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what is central dogma?






Replication:

The self-replication mechanism proposed by Watson and Crick, later termed semi-conservative replication means the first double-stranded DNA separates into two single strands and build two new DNA strand from the first ds DNA. The original statement is 
"It has not escaped our notice that the specific pairing we have postulate immediately suggests a possible copying mechanism for the genetic material"(Watson and crick, 1953).
 
How replication are accorded?

here is a simplified flowchart of DNA replication in prokaryotes:

Initiation:
  • Replication begins at the origin of replication (oriC).
  • The DNA double helix is unwound by the (enzyme helicase), creating a replication fork.
  • Single-strand binding proteins (SSBs) stabilize the unwound strands.
Elongation:
  • DNA polymerase synthesizes new DNA strands by adding nucleotides in the 5' to 3' direction, using the existing strands as templates.
  • The leading strand is synthesized continuously in the same direction as the replication fork movement.
  • The lagging strand is synthesized discontinuously in short fragments called Okazaki fragments, which are later joined by DNA ligase.
  • Primase synthesizes short RNA primers on both strands to initiate DNA synthesis.
Termination:
  • Replication is terminated when the replication forks meet at the termination region (ter).
  • DNA topoisomerase helps to relieve the tension in the DNA ahead of the replication fork.
  • The two daughter DNA molecules separate from each other.

Here is the flowchart:




Fig. replication process 



Here is a flowchart of the replication:

 Initiation
 Unwinding of DNA
  
 Stabilization of unwound strands by SSBs
  
 
 Synthesis of RNA primers by primase
  
 
 Elongation of leading and lagging strands by DNA polymerase
  
 
 Synthesis of Okazaki fragments on the lagging strand
  
 
 Joining of Okazaki fragments by DNA ligase
  
 
 Termination at the termination region (ter)
  
 
Separation of daughter DNA molecules Note that this flowchart is simplified and there are many more details and proteins involved in DNA replication in prokaryotes [E.coli].


Transcription:


The promoter is the DNA sequence where RNA polymerase binds to initiate transcription of the structural gene, while the terminator is the DNA sequence where transcription of the gene ends. The structural gene is the DNA sequence that codes for a particular protein or RNA molecule.

Transcription is the process by which DNA is used as a template to synthesize RNA. It begins when RNA polymerase, a multi-subunit enzyme, recognizes and binds to the promoter region of the DNA. The promoter contains specific DNA sequences that serve as recognition sites for DNA dependent-  RNA polymerase. Once RNA polymerase has bound to the promoter, it unwinds the DNA double helix and forms an open complex.

During elongation, RNA polymerase moves along the template strand of the DNA in the 3' to 5' direction, adding complementary nucleotides to the growing RNA chain in the 5' to 3' direction. As the RNA polymerase moves along the DNA template (or Template strand), it synthesizes RNA in a process that is sometimes referred to as a "single-stranded DNA template-dependent RNA polymerization." The RNA molecule grows in length, and the RNA polymerase moves along the DNA strand (referred to as the coding strand), unwinding it and re-forming the double helix behind it.

To explain the point a hypothetical sequence from a transcription unit is represented below:

3’-ATGCATGCATGCATGCATGCATGCATGCATGC-5’

5’-TACGTACGTACGTACGTACGTACGTACGTACG-3’

Termination occurs when RNA polymerase reaches a terminator sequence in the DNA, which signals the end of the gene being transcribed. The RNA polymerase, the newly synthesized RNA, and the DNA template dissociate from each other. The RNA transcript is released and can undergo further processing, such as splicing or modification of the 5' and 3' ends.

In prokaryotes, transcription is a tightly regulated process, with the rate and timing of transcription often controlled by environmental signals and cellular factors. Transcription is a critical step in gene expression, allowing the cell to synthesize the RNA molecules it needs to carry out various cellular functions.

Here is a flowchart of transcription:

 

Initiation:

  

 Binding of RNA polymerase to the promoter region of DNA

 

 Formation of open complex

  

 RNA polymerase initiates transcription at the start site

 

 Elongation:

 RNA polymerase moves along the DNA template, synthesizing RNA

 

 RNA polymerase unwinds and re-forms the DNA double helix

 

 Termination:

 RNA polymerase reaches the terminator sequence

RNA transcript is released

 

RNA undergoes further processing, such as splicing (breaking) or modification

Transcription


Note that there are many additional factors and proteins involved in prokaryotic transcription, such as sigma factors that help direct RNA polymerase to specific genes and transcription factors that regulate gene expression.


Translation:

DNA translation is the process by which the genetic information encoded in messenger RNA (mRNA) is used to synthesize a protein. This process occurs on ribosomes, which are composed of ribosomal RNA (rRNA) and proteins. 

Initiation:
The small subunit of the ribosome binds to the mRNA molecule near the start codon, which is usually AUG.
The initiator tRNA, which carries the amino acid methionine, recognizes and binds to the start codon with the help of initiation factors.
The large ribosomal subunit then binds to the complex, forming a complete ribosome and positioning the initiator tRNA in the P site of the ribosome.

Elongation:
A second tRNA carrying the appropriate amino acid binds to the A site of the ribosome.
Peptide bond formation occurs between the amino acid carried by the tRNA in the A site and the growing polypeptide chain attached to the tRNA in the P site.
The ribosome moves along the mRNA molecule in the 5' to 3' direction, shifting the tRNAs from the A and P sites to the P and E sites, respectively.
The empty tRNA in the E site is released from the ribosome and can be recharged with another amino acid.

Termination:
The ribosome reaches a stop codon in the mRNA, which does not code for an amino acid but signals the end of the protein coding sequence.
Release factors bind to the ribosome, causing it to release the completed polypeptide chain and the tRNA molecules.
The ribosome subunits dissociate from each other, and the mRNA molecule is released.
During translation, the genetic information encoded in the mRNA is decoded by the ribosome, which uses the information to synthesize a protein. The ribosome moves along the mRNA molecule in a 5' to 3' direction, adding amino acids to the growing polypeptide chain. Once a stop codon is reached, the ribosome releases the completed protein.

Overall, translation is a critical step in gene expression in prokaryotes, as it allows the cell to synthesize the proteins it needs to carry out various cellular functions.

Here is a flowchart of the translation:

Initiation:
Small ribosomal subunit binds to mRNA near start codon
Initiator tRNA recognizes and binds to start codon with help of initiation factors
Large ribosomal subunit binds to the complex, positioning initiator tRNA in P site

Elongation:
Second tRNA carrying appropriate amino acid binds to A site
Peptide bond formation occurs between amino acid in A site and growing polypeptide chain in P site
Ribosome moves along mRNA, shifting tRNAs from A/P sites to P/E sites
Empty tRNA in E site is released and can be recharged with another amino acid

Termination:
Ribosome reaches stop codon in mRNA
Release factors bind to ribosome, causing it to release completed polypeptide chain and tRNA molecules
Ribosome subunits dissociate from each other, and mRNA molecule is released.


Translation






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