Gene Expression of Bacteria
Here we are going to study of Mechanism of Prokaryotic DNA Transcription. Transcription is the first step in gene expression by which the genetic information in a strand of DNA is copied into a new molecule of messenger RNA (mRNA).
This process is known as the ‘Gene Expression’. This may also know as the central dogma of molecular biology. In order that express characters the genetic information (gene sequences) should be converted to proteins. For this first the nucleotide sequences should be transcribed into mRNA from DNA. And then the nucleotide sequence of mRNA should be translated into amino acids sequences to make proteins.
RNA is also a polymer of ribonucleotides. It is composed of ribose sugar molecule, A, G, C, U nitrogenous base pairs and phosphate group. In ribonucleotides the -OH group is in the 2nd carbon. RNA nucleotides are also held together by phosphates in the join the 3̍ C to the 5̍ C in the other. So, it is also having a 5̍-phosphate terminal and 3̍-hydroxyl terminals. RNA is single stranded. But it may have structural properties.
Single structure of nucleotides in RNA.
These single strands can fold up on itself to form double stranded regions.
3) Tertiary Structure
The secondary structure of nucleotide sequences are again fold and rap around itself to form tertiary structures. Double stranded RNA regions which are formed during secondary structure formation are rigid than the single stranded RNA regions. So, these regions can interact with single stranded regions again to form tertiary structures.
Different types of RNA
- mRNA (messenger RNA)
Copies the genetic information in DNA by complementary base pairing and carries this message to the ribosomes where the proteins are synthesized.
- tRNA (transfer RNA)
Picks the specific amino acid and transfers the amino acids to the ribosome and insert the correct amino acid into the chain according to the message written in the mRNA.
- rRNA (ribosomal RNA)
rRNA helps for the synthesis of ribosomes and proteins.
Mechanism of DNA Transcription
RNA polymerase initiates the synthesis of mRNA where ribonucleotides are added to the 3̍-OH ends of the growing RNA chain. RNA polymerase makes a complimentary copy of the DNA template. RNA polymerases do not need a pre-existing primer to initiate the synthesis of a new chain of RNA. Unlike DNA polymerase RNA polymerase does not require helicases to separate the strands. RNA polymerase enzyme consists of 2 components.
- Core Enzyme – Core enzyme have 5 subunits. Core enzyme itself cannot initiate the transcription.
- Holo Enzyme – The core enzyme with the s factor is called as the ‘Holo Enzyme’. Holo enzyme can initiate transcription.
There are 4 stages in transcription.
- Promoter recognition
- Initiation/ Chain initiation
- Chain elongation
- Chain termination
Sequence of DNA which encodes for a polypeptide chain or functional RNA. RNA transcripts are copied only from selective regions of the DNA (gene).
RNA polymerase holo enzyme can only initiate RNA synthesis at certain sites from a Ds DNA. These sites are known as ‘Promoters’.
Promoter sequence has 2 conserved sequences.
- -10 sequence – This is a short A-T rich region about 10 base pairs upstream of the transcription start site. Consensus sequence 5̍-TATAAT-3̍ (coding strand).
- -35 sequence – About 35 base pairs upstream of the start site. Consensus sequence of 5̍-TTGACA-3̍ (coding strand).
Transcription initiate site (+1) contains a purring nucleotide (A/G). RNA polymerase holo enzymes can recognize different types of promoters. Which promoter it can recognize is determined by the type of s factor. The most common promoters are those recognize by the RNA polymerase s70 in E. coli. RNA polymerase core enzyme binds to a s factor to form the holo enzyme and then the bound s factor directs the complex to the correct promoters.
The RNA polymerase recognizes the transcription start site and the 1st nucleotide is added at this site. RNA polymerase moves along the template DNA adding nucleotides to the 3̍-OH ends of the growing RNA.
The strands of DNA are opened at the protomer and the opening on the DNA helix is approximately 1.7 bp long. And it is called the ‘Transcription Bubble’. While the RNA polymerase moves along the DNA template polymerizing the RNA chain it forms a DNA-RNA hybrid of » 8-9 bp. The resulting RNA transcript emerges from the RNA polymerase through a channel and DNA strands rebind to each other. The RNA strand elongates until RNA polymerase reaches a termination sequence. When it reaches a termination sequence both the newly synthesized RNA transcribed and RNA polymerase will be released. Once the initiation is done s factor is released from RNA polymerase enzyme.
DNA Transcription Termination
There are two types of DNA transcription termination is known in prokaryotes.
- Factor independent
Only termination is depending only upon a specific DNA sequence.
- Factor dependent
Needs a specific sequence and an external factor or a protein.
- Factor independent method
The factor independent termination sites can be easily recognized because of the presence of 2 main parts.
- Inverted repeat sequence
- Short string of A in the template strand
The inverted repeat sequence (40 bp) is high in G-C content and this will be transcribed in to RNA. Due to its complimentary nature it can form a hairpin structure. The inverted repeat sequence is followed by a short string of A’s in the template strand (more than 6 A’s). This results in synthesis of a series of U in the mRNA strand. G-C rich hairpin structure of the mRNA may push the 3̍ end of the transcript from the active site. This is called ‘Back-tracking’. So, the less stable A and U base pairs in the DNA-RNA hybrid cause the separation of the transcript from the complex. Due to both these RNA transcript and RNA polymerase will dissociate from the complex terminating the transcription.
2. Factor dependent method
Factor dependent termination sequence are not readily recognizable. The important feature of this termination mechanism is it involves an external factor/protein to recognize the termination site. The major termination factor that has been recognized in E. coli is Rho (ρ). Rho (ρ) is an RNA dependent ATPase. It cleaves ATP to get energy in the presence of RNA. ρ is also a DNA-RNA helicase. It can separate only a double helix with RNA in one strand and DNA as the other strand. The ρ protein binds to a specific sequence present in the RNA known as ‘Rho Utilization’ (rut).
Once bound to the rut site ρ moves along the RNA in the 5̍®3̍ direction chasing the RNA polymerase. The energy for the movement of ρ is provided by the ATPase activity of the protein. When the RNA polymerase reaches the termination sequence the movement of RNA polymerase will be slow down. So, ρ factor can catch up the RNA polymerase and the RNA-DNA helicase activity of ρ protein can stop the transcription by releasing the RNA polymerase and mRNA transcript.
T.A.BROWN. (2010). GENE CLONING & DNA ANALYSIS (6th ed.). A John Wiley & Sons, Ltd,Publication.
Schleif, R. (2015). Genetics and Molecular Biology (2nd ed.). The Johns Hopkins University Press Baltimore and London.
Pasindu Chamikara – Microbiologist