MOLECULAR GENETICS 10

 Process of transcription

In prokaryotes

Transcription takes place in three steps

• Initiation
• RNA polymerase binds to promoter and initiates transcription.
• Initiation factor or sigma (σ) recognizes the promoter of the DNA.
• Elongation
• RNA polymerase facilitates opening of the helix and continues elongation.
• RNA polymerase uses nucleoside triphosphates as substrate and polymerizes in a template depended fashion following the rule of complementarity.
• Only a short stretch of RNA remains bound to the enzyme.
• Termination
• Once the polymerases reaches the terminator region RNA polymerase binds with the termination-factor (ρ) to terminate transcription.
• The nascent RNA falls off with the RNA polymerase which results in termination of transcription.

• The transcription and translation can be coupled in bacteria as the mRNA does not require any processing to become active, 
• Since only exons are there.
• and also transcription and translation take place in the same compartment

In eukaryotes

• There are two additional complexities in eukaryotes.

1. The first complexity is that there are at least three RNA polymerases in the nucleus.

• The RNA polymerase I transcribes rRNAs (28S, 18S, and 5.8S)
• The RNA polymerase III is responsible for transcription of tRNA, 5srRNA, and snRNAs (small nuclear RNAs).
• The RNA polymerase II transcribes precursor of mRNA, the heterogeneous nuclear RNA (hnRNA).

1. The second complexity is that the primary transcripts contain both the exons and the introns and are non-functional.
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• Primary transcripts are subjected to a process called splicing where the introns are removed and exons are joined in a defined order.

• hnRNA undergo two additional processing called as capping and tailing.
• In capping an unusual nucleotide (methyl guanosine triphosphate) is added to the 5'-end of hnRNA.
• In tailing, adenylate residues (200-300) are added at 3'-end in a template independent manner and the fully processed hnRNA is called mRNA

• mRNA is transported out of the nucleus for translation.

Significance of complexities

• The split-gene arrangements represent probably an ancient feature of the genome.
• The presence of introns is reminiscent of antiquity, and the process of splicing represents the dominance of RNA-world.

Genetic code

• The sequence of nucleotides on DNA which determines the sequence of amino acids in a polypeptide chain is termed as Genetic code.

• The process of translation requires transfer of genetic information from a polymer of nucleotides to a polymer of amino acids
•  but there is no complementarity between nucleotides and amino acids which led to the proposition of a genetic code that could direct the sequence of amino acids during synthesis of proteins.

(Minimum 20 amino acids,

4 bases,

1 bases /codon - only 4 amino acids

2 bases/ codon- combinations- 16 amino acids

So, 3 bases / codon - 64 combinations - easily code 20 proteins) -- means codon - triplet

• The salient features of genetic code are as follows:

• The codon is triplet, 61 codons code for amino acids and 3 codons do not code for any amino acids, hence they function as stop codons. (UAG, UAA, UGA)
• One codon codes for only one amino acid thus it is unambiguous and specific.

• Some amino acids are coded by more than one codon, hence the code is degenerate.

• The codon is read in mRNA in a contiguous fashion, there are no punctuations.

• The code is nearly universal. For example, from bacteria to human UUU would code for Phenylalanine (phe).

• AUG has dual functions, it codes for Methionine (met) , and it also act as initiator codon.

Mutation and genetic code

• Point mutation is the insertion or deletion of a single gene in the structural gene.
• 2 types - missense mutation - wrong amino acid added
• Nonsense mutation - stop codon is formed so peptide chain stopped here.

Example- point mutation is a change of single base pair in the gene for beta globin chain that results in the change of amino acid residue glutamate to valine, which results into a diseased condition called as sickle cell anaemia.

• Frame shift mutation is the insertion and deletion of three or its multiple bases which insert or delete one or multiple codon hence one or multiple amino acids, and reading frame remains unaltered from that point onwards.

Example- cystic fibrosis.

MOLECULAR GENETICS 9

 Transcription

• The process of copying genetic information from one strand of the DNA into RNA is termed as transcription.

• In transcription only a segment of DNA and only one of the strands is copied into RNA because
• if both strands act a template, they would code for RNA molecule with different sequences and the sequences of amino acids in the coded protein would be different.
• the two RNA molecules would be complementary to each other and would form a double stranded RNA which would prevent translation.

 

Transcription unit

A  transcription unit consists of

• A Promoter
• The Structural gene
• A Terminator

• The two strands of the DNA in the structural gene of a transcription unit is termed as template strand and coding strand.
• The strand that has the polarity 3'→5' acts as a template, and is referred as template strand.

• The other strand which has the polarity (5'→3') is referred as coding strand.
• The promoter and terminator flank the structural gene in a transcription unit.
• The promoter is located towards 5'-end (upstream) of the structural gene which provides binding site for RNA polymerase.
• The terminator is located towards 3'-end (downstream) of the coding strand which defines the end of the process of transcription.

Structure of a gene

• A gene is defined as the functional unit of inheritance.
• A gene also referred as a cistron can be defined as a segment of DNA coding for a polypeptide.
• The structural gene in a transcription unit could be said as monocistronic mostly in eukaryotes or polycistronic mostly in bacteria or prokaryotes.

• Exons are the coding sequences or expressed sequences that appear in mature or processed RNA.
• Introns are the intervening sequences which interrupt exons and do not appear in mature or processed RNA.

Types of RNA

• There are three major types of RNAs
• mRNA (messenger RNA),
• tRNA (transfer RNA),
• rRNA (ribosomal RNA).

 

• All three RNAs are needed to synthesize a protein in a cell.
• The mRNA provides the template, tRNA brings amino acids and reads the genetic code, and rRNAs play structural and catalytic role during translation

Structure of tRNA

• The tRNA, also called as sRNA (soluble RNA) has a role as an adapter molecule.

• tRNA has an anticodon loop that has bases complementary to the code.

• It has an amino acid accepter end to which it binds to amino acids.
• The secondary structure of tRNA looks like a clover-leaf.
• In actual structure, the tRNA is compact molecule which looks like inverted L.

rRNA-

MOLECULAR GENETICS 8

 Machinery & Enzymes for Replication

Enzymes play an important role acting as catalysts during the process of DNA replication. Some of the important enzymes are:

• DNA polymerase
• Helicase
• Primase
• DNA ligase

Energy source is needed to provide energy during the replication process.

 Deoxyribonucleoside triphosphates act as substrates & provide energy for polymerization reaction.

DNA polymerase

DNA polymerase creates DNA from nucleotides.

Also known as DNA dependent DNA polymerase

 It reads the existing DNA strands to create two new strands that match the existing ones. 

This enzyme is needed every time a cell divides so that one copy of DNA can be passed to each daughter cell.

DNA polymerase is a highly efficient enzyme, as it can replicate a large number of base pairs in a very short time. 

Rate of replication or Rate of polymerization is approx 2000 bp per second. 

A total of 4.6 * 106 base pairs are replicated within 18 minutes.  

DNA polymerase also catalyze with high degree of accuracy. A mistake is made once in every 1 billion base pairs copied. 

DNA polymerase proof reads to check for errors. However, these errors if remain can cause mutations.

Helicase

Enzyme Helicase unwinds DNA from tightly double stranded structure.

 Only after the strands are separated, DNA polymerase can do its job of creating the new strands. 

This enzyme separates the strands by breaking the hydrogen bonds between the bases of the two strands.

Primase

This enzyme creates a short fragment of RNA (primer) paired with the template DNA strand. 

This enzyme initiates the process of creation of new strands. 

DNA polymerase cannot initiate the process on its own. Therefore, primase initiates the same

Ligases

It binds various small fragments of DNA.

Process of DNA replication

Replication cannot be initiated in any random part of DNA. 

Region in a DNA where replication initiates is termed as ‘Origin of Replication’. (Ori)

Prokaryotes- usually 1 ori

Eukaryotes- multiple

Step 1.

Enzyme Helicase breaks hydrogen bonds, thus separating the two strands of DNA. Replication fork structure is formed.

Step 2.

New strand formation- only in one direction 5' - 3'

1. Continuous synthesis takes place in the Leading strand. (3' -5') 
2.  In this strand, DNA is synthesized in the same direction as the growing replication fork. 

1. Discontinuous synthesis takes place in the Lagging strand. 
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3. DNA polymerase can add new free nucleotides to the 3’ end of the new strand. In the lagging strand, no free 3’-OH end is available. 
4. Therefore, DNA polymerase is unable to initiate the process. 
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6. Enzyme Primase initiate the process by creating a small RNA fragment called Primer. 
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8. DNA polymerase then extends the primed segments adding free nucleotides. 
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10. RNA primers are replaced with DNA.
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12.  Thus, we have DNA fragments. direction of synthesis in Lagging strand (It is opposite to the direction of growing replication fork). DNA Ligase now joins the DNA fragments and forms a complete DNA.

These DNA fragments are termed as ‘Okazaki fragments’ after the name of the scientist who first described the process of Discontinuous synthesis on Lagging strand.

Later, fragments are joined by Ligase enzyme.

This entire process of DNA replication occurs during S-phase of cell cycle in eukaryotes. Research is still going on for more detail on the replication process.

MOLECULAR GENETICS 7

 Meselson-Stahl Experiment

This experiment was performed to prove the semi conservative nature of DNA replication.  

On bacteria E.coli in 1958.

Basis of the Experiment

1. If E.coli was grown in a medium with N-15 (isotope of Nitrogen), the E.coli had DNA with  N-15 isotope.

If E.coli was grown in a medium with N-14 (more abundant isotope of Nitrogen), the E.coli had DNA with N-14 isotope.

It was then observed with Centrifugation that DNA with N-15 is heavier than that of N-14.

Making use of the fact that DNA with N-15 is heavier than DNA with N-14, this experiment was performed.

Experiment-

Step 1.  E.Coli was grown in a medium with N-15 for several generations -- all DNA have N15

Step 2.  E.coli with only N-15 in their DNA were transferred to a medium with N-14

Cells of E.coli were allowed to divide. 

Sample was taken and DNA was extracted periodically as cell division continued to check what type of DNA is being formed now. 

One replication in E.coli takes around 20 minutes. So, generation I is formed in 20 minutes.

Therefore samples are taken after 20 minutes, then again after 40 minutes. 

Densities of DNA from the sample were measured to reach to results & conclusion.

Results

Generation I: DNA was found to have intermediate density after 1 replicati

Generation II: Equal amounts of DNA with two different densities were found

Conclusion

• Presence of a hybrid/ intermediate density excluded Conservative hypothesis. Had it been Conservative hypothesis, Generation 1 would have been either Blue(N-15) or Green(N-14); and not an Intermediate one.

• Presence of N-14 DNA in Generation II excluded Dispersive hypothesis. If it was Dispersive, each DNA should have had the same density. But, in Generation II, we could see 50% of the DNA have intermediate density, whereas remaining 50% have N-14 density.

• Semi-conservative hypothesis could explain the entire experimental result. Separation of strands concept could explain the outcomes of Generation I & II.

Thus, it was proved that DNA replication is Semi-conservative in nature.

MOLECULAR GENETICS 6

 Central Dogma of Molecular Biology

Francis Crick in 1956 proposed the hypothesis of Central Dogma This explains the flow of genetic information in any biological system

Three major classes of biopolymers are involved in this flow: DNA, RNA and Proteins.

With these 3 categories of biopolymers, a total of 9 transfers can be possible which are grouped under the following categories:

1. General transfers: These transfers occur in most of the organisms
2. a) DNA -> DNA (Replication)
3.        b) DNA -> RNA (Transcription)
4.        c) RNA -> Proteins (Translation)

In this lesson, we will discuss about these General transfers in detail.

2. Special transfers: These occur in viruses where RNA is the genetic material
3. a) RNA -> RNA
4.         b) RNA -> DNA
5.         c) DNA -> Proteins
6. Unknown transfers: These transfers might be possible but yet not known
7. a) RNA -> RNA
8.     b) RNA -> DNA

       c) DNA -> Proteins

DNA Replication

Replication is the process of reproducing or creating a copy of something. In this topic, we will see how DNA creates a copy of itself.

Various hypotheses were proposed by various scientists regarding the replication model of DNA i.e. how DNA replicates. Some of these were:

• Semi-conservative DNA replication model
• Conservative DNA replication
• Dispersive DNA replication

 

Semi-conservative DNA replication model

Watson & Crick suggested the ‘Semi conservative DNA replication’ model. According to this model, the two strands of DNA separate.

Each strand act as template for synthesis of a new strand. The new strand is synthesized based on complementary base pairing with the template.

Each new DNA molecule = 1 parental strand & 1 one newly formed strand. 

This is how the original DNA molecule (1 copy) gives rise to two copies.

Conservative DNA replication

According to this model, Complete DNA molecule (and not just one strand) acts as a template for new synthesis.

Dispersive DNA replication

According to the Dispersive model, the new DNA is synthesized in short pieces. A part of the old strand is attached to the end of a part of newly synthesized strand.