Fundamentals of Microbiology

Lesson 22. DNA REPLICATION, TRANSCRIPTION, TRANSLATION

Module 6. Bacterial genetics

Lesson 22

DNA REPLICATION, TRANSCRIPTION, TRANSLATION

22.1 Introduction

One major question for the human mind is how life continues. One of the most important mechanisms for all life cells to give off springs is undoubtedly the DNA Replication. DNA Replication answers to the question: ‘When a cell divides, where the extra DNA comes from?’ What ‘DNA Replication’ is? It is the process that can duplicate the DNA of a cell. The next step is the cell to duplicate. Every cell (of eukaryotes or prokaryotes) has one or more DNA (or RNA) polymer molecules that need to duplicate in order the cell duplication to take place. This is what DNA replication or DNA synthesis succeeds. In the eukaryotes (organisms with cell that have nucleus) the DNA is formed in two strands, each composed of units called nucleotides. The two strands look like two chains that form the DNA double helix. The DNA replication process is capable of opening the double helix and separating the two strands. Then the two strands are copied. As a result two new DNA molecules are created. The next step is the cell division. After that a daughter cell is created. In its nucleus lies a copy of the parental DNA.

22.2 DNA Replication Models

The process of DNA Replication was hiding many secrets. One of the most important was how the two daughter strands are created. In order the hereditary phenomenon to be explained, these strands should be accurately copied and transmitted from the parental cell to the daughter ones. These are three possible models that describe the accurate creation of the daughter chains.

22.2.1 Semiconservative replication

According to this model, DNA Replication would create two molecules. Each of them would be a complex of an old (parental and a daughter strand).

22.2.2 Conservative replication

According to this model, the DNA Replication process would create a brand new DNA double helix made of two daughter strands while the parental chains would stay together.

22.2.3 Dispersive replication

According to this model the replication process would create two DNA double-chains, each of them with parts of both parent and daughter molecules.

22.3 Steps in DNA Replication

1) The first major step in DNA Replication to take place is the breaking of hydrogen bonds between bases of the two anti-parallel strands. The un-wounding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T. That is because there are only two bonds between Adenine and Thymine (there are three hydrogen bonds between Cytosine and Guanine). Helicase is the enzyme that splits the two strands. The initiation point where the splitting starts is called ‘origin of replication’.The structure that is created is known as ‘Replication Fork’ (Fig. 22.1).

Fig. 22.1 Replication fork

2) One of the most important steps of DNA Replication is the binding of RNA Primase in the the initiation point of the 3'-5' parent chain. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides (Fig. 22.2).

Fig. 22.2 Binding of RNA primase

3) The elongation process is different for the 5'-3' and 3'-5' template.

a) 5'-3' Template: The 3'-5' proceeding daughter strand -that uses a 5'-3' template- is called leading strand because DNA Polymerase A can read the template and continuously adds nucleotides (complementary to the nucleotides of the template, for example Adenine opposite to Thymine etc.) (Fig.22.3).

Fig. 22.3 Formation of leading and lagging strands

b) 3'-5'Template: The 3'-5' template cannot be read by DNA Polymerase. The replication of this template is complicated and the new strand is called lagging strand. In the lagging strand the RNA primase adds more RNA primers. DNA polymerase A reads the template and lengthens the bursts. The gap between two RNA primers is called ‘Okazaki Fragments’. The RNA primers are necessary for DNA polymerase A to bind nucleotides to the 3' end of them. The daughter strand is elongated with the binding of more DNA nucleotides (Fig. 22.4).

Fig. 22.4 Okazaki fragments

4) In the lagging strand the DNA Pol I - exonuclease reads the fragments and removes the RNA primers. The gaps are closed with the action of DNA Polymerase (adds complementary nucleotides to the gaps) and DNA Ligase (adds phosphate in the remaining gaps of the phosphate - sugar backbone).
Each new double helix is consisted of one old and one new chain. This is what we call semi conservative replication (Fig.22.5)

Fig. 22.5 Semi conservative replication-removal of primers

5) The last step of DNA replication is the termination. This process happens when the DNA Polymerase reaches to an end of the strands (Fig. 22.6) We can easily understand that in the last section of the lagging strand, when the RNA primer is removed, it is not possible for the DNA Polymerase to seal the gap (because there is no primer). So, the end of the parental strand where the last primer binds isn't replicated.

6) The DNA Replication is not completed before a mechanism of repair fixes possible errors caused during the replication. Enzymes like nucleases remove the wrong nucleotides and the DNA polymerase fills the gaps.

Fig. 22.6 DNA strands

22.4 Transcription

Transcription is the mechanism by which a template strand of DNA is utilized by specific RNA polymerases to generate one of the three different classifications of RNA. These 3 RNA classes are:

• Messenger RNAs (mRNAs): This class of RNAs is the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation.
• Transfer RNAs (tRNAs): This class of small RNAs form covalent attachments to individual amino acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain.
• Ribosomal RNAs (rRNAs): This class of RNAs is assembled, together with numerous ribosomal proteins, to form the ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide synthesis.

All RNA polymerases are dependent upon a DNA template in order to synthesize RNA. The resultant RNA is, therefore, complimentary to the template strand of the DNA duplex and identical to the non-template strand. The non-template strand is called the coding strand because its' sequences are identical to those of the mRNA. However, in RNA, U is substituted for T.

22.5 Translation

Translation is the RNA directed synthesis of polypeptides. This process requires all three classes of RNA. Although the chemistry of peptide bond formation is relatively simple, the processes leading to the ability to form a peptide bond are exceedingly complex. The template for correct addition of individual amino acids is the mRNA, yet both tRNAs and rRNAs are involved in the process. The tRNAs carry activated amino acids into the ribosome which is composed of rRNA and ribosomal proteins. The ribosome is associated with the mRNA ensuring correct access of activated tRNAs and containing the necessary enzymatic activities to catalyze peptide bond formation.

22.5.1 The genetic code

Shown below are the triplets that are used for each of the 20 amino acids found in eukaryotic proteins. The row on the left side indicates the first nucleotide of each triplet and the row across the top represents the second nucleotide. The wobble position nucleotides are indicated in blue. The three stop codons are highlighted in red (Fig. 22.7).

Fig. 22.7 Triplet codons

22.5.2 Order of events in translation

The ability to begin to identify the roles of the various ribosomal proteins in the processes of ribosome assembly and translation was aided by the discovery that the ribosomal subunits will self assemble in vitro from their constituent parts (Fig. 22.8).

Following assembly of both the small and large subunits onto the mRNA, and given the presence of charged tRNAs, protein synthesis can take place. To reiterate the process of protein synthesis:

• Synthesis proceeds from the N-terminus to the C-terminus of the protein.
• The ribosomes read the mRNA in the 5' to 3' direction.
• Active translation occurs on polyribosomes (also termed polysomes). This means that more than one ribosome can be bound to and translate a given mRNA at any one time.
• Chain elongation occurs by sequential addition of amino acids to the C-terminal end of the ribosome bound polypeptide.

Translation proceeds in an ordered process. First accurate and efficient initiation occurs, then chain elongation, and finally accurate and efficient termination must occur. All three of these processes require specific proteins, some of which are ribosome associated and some of which are separate from the ribosome, but may be temporarily associated with it.
Initiation

• First the 70s ribosome must separate into 50s and 30s subunits.
• The 3 initiation factors IF-1, IF-2, and IF-3 then bind to the 30S ribosomal subunit.
• Then the specific initiator tRNA carrying the N-formylmethionine and the mRNA join the complex (remember base pairing via Shine-Dalgarno sequence). The initiator tRNA is specifically recognized by IF-2.
• At this point in the process we have what is termed a 30s preinitiation complex.
• The 50S subunit binds, triggering the release of the IFs.

Thus, formation of the 70S initiation complex is completed and it is ready for the next phase, elongation.

Fig. 22.8 Order of events in translation