Archive for September, 2007

To qualify as the genetic material, DNA has to fulfil two key requirements:

1) Genotype function or Replication: The genetic material must have the capability to store genetic information, and transmit this information faithfully from parents to offspring, generation after generation. This indicates that the genetic material should have the ability to replicate itself and make its copies.

2) Phenotype function or Gene Expression: The genetic material must control the development of phenotype of the organism. It means that it should control the growth and differentiation of the organism from the single-celled zygote to the mature adult.

There are three landmark experiments, which showed that DNA is the genetic material.

1) Griffith’s Experiment (1928)

2)  Avery, MacLeod and McCraty’s Experiment (1944): They showed that if highly purified DNA from Type IIIS pneumococci was present with TypeIIR pneumococci, some of the pneumococci were transformed to Type IIIS.

3)  Hershey-Chase Experiment (1952): The basis for the experiment: DNA contains phosphorus but no sulfur; whereas proteins contain sulfur but no phosphorus. The experiment is also referred to as the Waring blender experiment. Alfred D. Hershey won the Noble Prize in Physiology or Medicine in 1969.

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I know this is very elementary and any high school student must be knowing the differences between RNA and DNA. But I feel a blog which deals with diverse aspects of molecular biology and biotechnology deserves a mention of basic differences between the two molecules.

DNA is double helical; RNA is single helical.

DNA contains deoxyribose pentose sugar (2′-deoxy-D-ribose); RNA has D-ribose sugar. The oyxgen from 2′ carbon of the pentose sugar is absent in deoxyribose sugar.

Both type of pentoses occur in their β-furanose (closed five-membered ring) form.

DNA, thus, consists of deoxyribonucleoside 5′-monophosphates (deoxyribonucleotides); RNA has ribonucleoside 5′-monophosphates (ribonucleotides).

DNA contains adenine (A), guanine (G), cytosine (C) and thymine (T) bases; RNA has adenine (A), guanine (G), cytosine (C) and uracil (U) bases. Thymine is not found in RNAs.

DNA is the genetic material in all organisms; however, RNA forms the genetic material in certain viruses like SARS, influenza, hepatitis C, retroviruses, etc.

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The base of a nucleotide is joined covalently in an N-β-glycosyl bond to the 1′ carbon of the pentose sugar, and the phosphate is esterified to 5′ carbon. The N-β-glycosyl bond is formed by removal of water, as in O-glycosidic bond formation. The nitrogenous bases present in nucleic acids are:

1) Purines: Adenine and guanine

2) Pyrimidines: Cytosine, thymine and uracil.

The main function of nucleic acids is the storage of information for all life processes and its expression.

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Nucleic acids (DNA and RNA) are the polymers of nucleotides. Each nucleotide is made up of 3 components: 1) a pentose sugar, 2) a phosphate, and 3) a nitrogenous (nitrogen-containing) base. A nucleotide without the phosphate group is called a nucleoside. The nitrogenous bases are derivatives of two compounds: pyrimidine and purine. The bases and pentose sugar are heterocyclic compounds. The carbons of the pentose sugar are given a prime (‘) designation to differentiate them from the numbered atoms of the nitrogenous bases.

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Ribosomal RNAs (rRNAs) constitute ribosomes where protein synthesis occurs. rRNA and ribosomal proteins combine to form ribosomes. Both prokaryotic and eukaryotic rRNA are made from longer precursors called preribosomal RNAs, or pre-rRNA .

In bacteria, a single 30S, 6500 nucleotides long, RNA precursor, after processing, makes 16S, 23S and 5S rRNAs. RNA at both ends of 30S precursor and between the rRNAs is removed during processing. The E. coli genome encodes 7 pre-rRNA molecules. All these genes have identical rRNA coding regions; but the regions in between the coding regions differ. The regions between the genes for 16S and 23S rRNA code for one or two tRNAs.

In eukaryotes, a 45S pre-rRNA transcript is processed to give rise to 18S, 28S and 5.8S rRNAs. The processing takes place in the nucleolus. The 5S rRNA is synthesized as a separate transcript.

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Poly(A) tail

At the 3′ end, most eukaryotic mRNAs have a string of 80-250 adenylate residues called the poly(A) tail. The poly(A) tail is added in a multistep process after the mRNA transcript has been synthesized. Two sequences are required for cleavage and polyadenylation (addition of ploy(A) tail) of the mRNA: 1) A highly conserved polyadenylation signal sequence AAUAAA, found 10 to 30 nucleotides upstream (on the 5′ side) of the cleavage; 2) A less well-defined sequence rich in G and U or U residues only, found 20 to 40 nucleotides downstream of the cleavage site. The cleavage generates the free 3′-OH group that defines the end of the mRNA, to which adenylate residues are added. Following proteins are required for cleavage and polyadenylation of pre-mRNAs.

1) Cleavage and polyadenylation specificity factor (CPSF): It is 360 kDa large complex made up of 4 polypeptides. It forms an unstable complex with the AAUAAA sequence.

2) Cleavage stimulatory factor (CStF1): 200 kDa heterotrimer.

3) Cleavage Factor I and II (CFI and CFII)

After CPSF has bound to the AAUAAA on the pre-mRNA, the CStF1, CFI, and CFII bind to the CPSF-mRNA complex. Interaction between CStF and GU or U-rich less well-defined sequence stabilizes the multiprotein complex. Finally, polyadenylate polymerase (poly(A) polymerase or PAP) binds to the complex before the cleavage can occur. The PAP binding links cleavage and polyadenylation, so that the free 3′ end generated after cleavage is rapidly polyadenylated. The assembly of this large multiprotein cleavage-polyadenylation complex around the AAUAAA signal in a pre-mRNA is analogous in many ways to the formation of the transcription-initiation complex at the TATA box.

Following cleavage at the poly(A) site, polyaenylation proceeds in two phases. Addition of the first 12 or so A residues occurs slowly, followed by a rapid addition of upto 200-250 A residues. The rapid phase requires the binding of multiple copies of poly(A) binding protein (PABII). PABII stimulates polymerization of additional A residues by PAP. PABII is also responsible for signaling PAP to terminate polymerization when te poly(A) tail reaches a length of 200-250 residues.

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The 5′ end of eukaryotic mRNA is capped with a guanine nucleotide (which is methylated forming 7-methyguanosine). The cap (5′-G) is added to the mRNA after transcription. The addition of 5′ G is catalyzed by a nuclear enzyme, guanylyl transferase. The cap is linked to the 5′ terminus of the mRNA through an unusual 5′,5′-triphospahe linkage. The 5′ cap is formed by condensation of a molecule of GTP with the triphosphate at the 5′ end of the transcript. The guanine is subsequently methylated at N-7 to form 7-methylguanosine. Additional methyl groups are added to the 2′ hydroxyls (-OH) of the first and second nucleotides adjacent to the cap. The methyl groups are derived from S-adenosylmethionine.

At the 3′ end, most eukaryotic mRNAs have a string of 80-250 adenylate residues called the poly(A) tail.

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