TRANSCRIPTION OF PROKARYOT MOLECULARES.pptx

TRANSCRIPTION

1. TRANSCRIPTION OF PROKARYOTES Presented By: Muhammad Kashif Nazir

2. INTRODUCTION Definition: A central event in gene expression is the copying of the sequence of the template strand of a gene into a complementary RNA transcript. All cells have at least one kind of RNA polymerase the enzyme that transcribes RNA from DNA and the machinery that translates the mRNA into protein. In bacteria, transcription and translation are said to be “coupled,” since they occur within a single cellular compartment. Transcription takes place in the cytoplasm in prokaryotes. The whole process of transcription and translation occurs within minutes.

3. RNA polymerase The bacterial RNA polymerase is comprised of a core enzyme plus a transcription factor called the sigma factor (σ). Together, they form the complete, fully functional enzyme complex called the holoenzyme . The core enzyme: The core enzyme is the component of the holoenzyme that catalyzes polymerization. It is 400 kD in size and has five subunits: two copies of the α-subunit ( αI , αII ), one copy each of the β-, β ′-, ω- subunits

4. Cofactor: The Sigma subunit (sigma factor) enables RNA pol to recognize promoter regions on the DNA. The Sigma subunit plus the core enzyme make up the holoenzyme . [Note: Different a factors recognize different groups of genes, with Sigma70 predominating.]

5. Stages Of Transcription Transcription is the synthesis of RNA from DNA. It is further divided in to 3 steps: Initiation    Elongation   Termination

6. INITIATION If nucleotide sequences of DNA are aligned with each other and each has exactly the same series of nucleotides in a given region, the sequence is said to be conserved. Transcription begins with the binding of the RNA pol holoenzyme to a region of the DNA known as the promoter, which is not transcribed . The prokaryotic promoter contains characteristic consensus sequences. It serves as start signal for RNA polymerase to start reaction.

7. The promoter consensus sequence consists of two hexamer sequences. The start of transcription by the number +1 and to use positive numbers to count farther down the DNA in the direction of transcription, the direction referred to as downstream (3′ to the start site ). If transcription is proceeding to the right, then the direction to the left (5 ′ to the start site) is called upstream with the bases indicated by negative numbers.

8. –35 Sequence: A consensus sequence (5-TTGACA-3), centered about 35 bases to the left of the transcription start site is the initial point of contact for the holoenzyme , and a closed complex is formed . Pribnow box: The holoenzyme moves and covers a second consensus sequence (5 -TATAAT-3), centered at about –10, which is the site of initial DNA melting (unwinding). Melting of a short stretch (about 14 bases) converts the closed complex to an open complex known as a transcription bubble.

9. Elongation: Once the promoter region has been recognized and bound by the holoenzyme , local unwinding of the DNA helix continues mediated by the polymerase. RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase is said to begin when the transcript (typically starting with a purine ) exceeds ten nucleotides in length. Sigma is then released, and the core enzyme is able to leave (“clear”) the promoter and move along the template strand.

10. During transcription, a short DNA–RNA hybrid helix is formed. Like DNA pol , RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5 →3 direction. In contrast to DNA pol , RNA pol does not require a primer and does not appear to have 3 →5 exonuclease (proofreading) activity.

11. Termination: The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (spontaneous) or dependent upon the participation of a protein known as the ρ (rho) factor. ρ-Independent termination : Seen with most prokaryotic genes, this requires that a sequence in the DNA template generates a sequence in the nascent (newly made) RNA that is self-complementary. This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by hydrogen bonds) plus a loop. This structure is known as a “hairpin .” Additionally , just beyond the hairpin, the RNA transcript contains a string of Us at the 3 -end. The bonding of these Us to the complementary as of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix “zips up” behind the RNA polymerase.

12. ρ -Dependent termination : This requires the participation of an additional protein, rho (r), which is a hexameric ATPase with helicase activity . Rho binds a C-rich “rho recognition site” near the 5 -end of the nascent RNA and, using its ATPase activity, moves along the RNA until it reaches the RNA pol paused at the termination site. The ATP-dependent helicase activity of separates the RNA– DNA hybrid helix, causing the release of the RNA.