Transcription in prokaryotes: Initiation, Elongation and Termination

Transcription: Initiation, Elongation and Termination

Introduction of Transcription

  • The synthesis of RNA under the direction of DNA is called transcription. The RNA product has a sequence complementary to the DNA template directing its synthesis.
  • Thymine is not normally found in mRNA and rRNA. Although adenine directs the incorporation of thymine during DNA replication, it usually codes for uracil during RNA synthesis.

RNA Bases Coded for by DNA

DNA Base Purine or Pyrimidine Incorporated into RNA
Adenine Uracil
Guanine Cytosine
Cytosine Guanine
Thymine Adenine
  • Transcription generates three kinds of RNA:
    • Messenger RNA (mRNA) bears the message for protein synthesis. In Bacteria and Archaea, the mRNA often bears coding information transcribed from adjacent genes. Therefore it is said to be polygenic or polycistronic.
A Polycistronic Bacterial Messenger RNA
    • Transfer RNA (tRNA) carries amino acids during protein synthesis.
    • ribosomal RNA (rRNA) molecules are components of ribosomes.

Transcription in Bacteria

  • RNA is synthesized under the direction of DNA by the enzyme RNA polymerase.
  • The reaction is quite similar to that catalysed by DNA polymerase. ATP, GTP, CTP, and UTP are used to produce an RNA complementary to the DNA template. As mentioned earlier, these nucleotides contain ribose rather than deoxyribose.
  • RNA synthesis, like DNA synthesis, proceeds in a 5′ to 3′ direction with new nucleotides being added to the 3′ end of the growing chain at a rate of about 40 nucleotides per second at 37°C. In both DNA and RNA polymerase reactions, pyrophosphate (PPi) is produced.
  • It is then hydrolyzed to orthophosphate in a reaction catalyzed by the pyrophosphatase enzyme. Hydrolysis of the pyrophosphate product makes DNA and RNA synthesis irreversible. If the pyrophosphate level were too high, DNA and RNA would be degraded by a reversal of the polymerase reactions.
  • RNA synthesis, like DNA synthesis, proceeds in a 5 to 3 direction with new nucleotides being added to the 3′ end of the growing chain at a rate of about 40 nucleotides per second at 37°C. In both DNA and RNA polymerase reactions, pyrophosphate (PPi) is produced.
  • It is then hydrolyzed to orthophosphate in a reaction catalyzed by the pyrophosphatase enzyme. Hydrolysis of the pyrophosphate product makes DNA and RNA synthesis irreversible. If the pyrophosphate level were too high, DNA and RNA would be degraded by a reversal of the polymerase reactions.
  • Most bacterial RNA polymerases contain five types of polypeptide chains: α, β, β′, ω, and σ. The core enzyme is composed of five chains α2, β, β′, ω, and σ) and catalyzes RNA synthesis.
  • The sigma factor (σ) has no catalytic activity but helps the core enzyme recognize the start of genes. When sigma is bound to core enzyme, the six-subunit complex is termed RNA polymerase holoenzyme.
  • Only holoenzyme can begin transcription, but as we will see, core enzyme completes RNA synthesis once it has been initiated. The precise functions of the α, β, β′, and ω polypeptides are not yet clear.
  • The α subunits seem to be involved in the assembly of the core enzyme, recognition of promoters, and interaction with some regulatory factors.
  • The binding site for DNA is on β′, and the ω subunit seems to be involved in stabilizing the conformation of the β′ subunit. The
    β subunit binds ribonucleotide substrates. Rifampin, an RNA polymerase inhibitor, binds to the β subunit.

Major events in Transcription

Each gene or set of genes contains a specific promoter region for guiding the beginning of transcription. This is followed by the region of the genes that is transcribed and ends with a terminator that stops transcription. DNA is unwound at the promoter by RNA polymerase. Only one strand of DNA, called the template strand, is used to guide RNA synthesis by the RNA polymerase. This strand runs in the 3′ to 5′ direction.
As the RNA polymerase moves along the strand, it adds complementary nucleotides as dictated by the DNA template, forming the single-stranded mRNA that reads in the 5′ to 3′ direction.
The polymerase continues transcribing until it reaches a termination site and the mRNA transcript is released for translation. Note that the section of the DNA that has been transcribed is rewound into its original configuration.

Transcription involves three separate processes

  1. Initiation
  2. Elongation
  3. Termination

Initiation

The sigma factor of the RNA polymerase holoenzyme is responsible for positioning the core enzyme properly at the promoter. Sigma factor recognizes two regions in the promoter, one centered at -35 and the other centered at -10. Once positioned properly, the DNA at the -10 region unwinds to form an open complex. The sigma factor dissociates from the core enzyme as it begins transcribing the gene.
  • Only a relatively short segment of DNA is transcribed (unlike replication in which the entire chromosome must be copied), and initiation begins when the RNA polymerase binds to the promoter for the gene. RNA polymerase core enzyme is not able to bind DNA tightly or specifically. This situation is drastically changed when sigma is bound to core to make the holoenzyme, which binds the promoter tightly.
  • The promoter serves only as a target for the binding of the RNA polymerase and is not transcribed. Bacterial promoters have two characteristic features: a sequence of six bases (often TTGACA) about 35 bases pairs before the transcription starting point and a TATAAT sequence or Pribnow box, usually about 10 base pairs upstream of the transcriptional start site.
  • These regions are called the -35 and -10 sites, respectively, while the first nucleotide to be transcribed is referred to as the +1 site. As noted previously, RNA polymerase holoenzyme recognizes the specific sequences at the -10 and -35 sites of promoters. Because the sites must be similar in all promoters, they are called consensus sequences.

Elongation

  • Once bound to the promoter site, RNA polymerase is able to unwind the DNA without the aid of helicases. The -10 site is rich in adenines and thymines, making it easier to break the hydrogen bonds that keep the DNA double stranded; when the DNA is unwound at this region, it is called open complex.
  • A region of unwound DNA equivalent to about two turns of the helix (about 16–20 bases pairs) becomes the “transcription bubble,” which moves with the RNA polymerase as it proceeds to transcribe mRNA from the template DNA strand during elongation. Within the transcription bubble, a temporary RNA: DNA hybrid is formed.
The transcription Bubble
  • As the RNA polymerase progresses in the 3′ to 5′ direction along the DNA template, the sigma factor soon dissociates from core RNA polymerase and is available to aid another unit of core enzyme initiate transcription. The mRNA is made in the 5′ to 3′ direction so it is complementary and antiparallel to the DNA template.
  • As elongation of the mRNA continues, single-stranded mRNAis released and the two strands of DNA behind the transcription bubble resume their double helical structure. RNA polymerase is a remarkable enzyme capable of several activities, including unwinding the
    DNA, moving along the template, and synthesizing RNA.

Termination

  • Termination of transcription occurs when the core RNA polymerase dissociates from the template DNA.
  • The end of a gene or group of genes is marked by DNA sequences in the trailer (which is transcribed but not translated) and the terminator.
  • The sequences within procaryotic terminators often contain nucleotides that, when transcribed into RNA, form hydrogen bonds within the single-stranded RNA. This intrastrand base pairing creates a hairpin-shaped loop-and-stem structure.
  • This structure appears to cause the RNA polymerase to pause or stop transcribing DNA.
  • There are two kinds of terminators.
    • The first type causes intrinsic or rho-independent termination: It features the mRNA hairpin followed by a stretch of about six uridine residues. Once the RNA polymerase has paused at the hairpin loop, the A-U base pairs in the uracil-rich region are too weak to hold the RNA:DNA duplex together and the RNA polymerase falls off.
This type of terminator contains a U-rich sequence downstream from a stretch of nucleotides that can form a stem-loop and stem structure. Formation of the stem loop in the newly synthesized RNA causes RNA polymerase to pause.This pausing is stabilized by the NusA protein. The U-A bonds in the uracil-rich region are not strong enough to hold the RNA and DNA together. Therefore, the RNA, DNA, and RNA polymerase dissociate and transcription stops.
    • The second kind of terminator lacks a poly-U region, and often the hairpin: It requires the aid of a special protein, the rho factor (Ρ). This terminator causes rho-dependent termination. It is thought that rho binds to mRNA and moves along the molecule until it reaches the RNA polymerase that has halted at a terminator. The rho factor, which has hybrid RNA:DNA helicase activity, then causes the polymerase to dissociate from the mRNA, probably by unwinding the mRNA-DNA complex.
Rho-Factor (P) -Dependent Termination of Transcription. The rut site stands for rho utilization site.

Reference and sources

  • https://open.oregonstate.education/molecular-biology/chapter/transcription-of-rna-and-its-modification/
  • https://en.wikipedia.org/wiki/Protein_biosynthesis
  • https://infoanew.com/wikipedia/RNA
  • https://www.researchgate.net/publication/6843167_Rho-dependent_terminators_and_transcription_termination
  • https://quizlet.com/ma/275572817/quiz-chapter-9-dna-to-proteins-flash-cards/
  • https://www.cram.com/flashcards/biol-351-test-2-897559
  • https://www.learninsta.com/ncert-solutions/ncert-exemplar-solutions-for-class-12-biology-chapter-6/
  • https://www.toppr.com/ask/question/choose-the-correct-answer-from-the-alternatives-given-thernapolymerase/
  • https://quizlet.com/169221055/microbiology-chapter-13-flash-cards/
  • https://www.studocu.com/en-au/document/university-of-technology-sydney/general-microbiology/pastexams/exam-november-2018-answers/6139286/view
  • https://www.preservearticles.com/biology/explain-the-process-of-transcription-in-prokaryotes/16271
  • https://quizlet.com/235744766/biol-202-unit-2-grqs-flash-cards/

Transcription: Initiation Elongation and Termination

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