You have created an in vitro transcription system which contains all the components for transcription including DNA with a bacterial transcription unit. You discover that transcription is initiated, but from random points on the DNA. Which of the following components in your system is most likely defective or missing to cause transcription to occur in this fashion?

Question

You have created an in vitro transcription system which contains all the components for transcription including DNA with a bacterial transcription unit.  You discover that transcription is initiated, but from random points on the DNA. Which of the following components in your system is most likely defective or missing to cause transcription to occur in this fashion?

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Question restatement: In your in vitro transcription system containing all components for transcription, including DNA with a bacterial transcription unit, transcription initiates but from random points on the DNA. Which component is most likely defective or missing to cause this pattern?

Option 1: sigma factor
This is the bacterial transcription initiation factor that directs RNA polymerase to specific promoter sequences by recognizing the -35 and -10 elements. Without sigma, the core RNA polymerase cannot efficiently locate promoters and tends to bind DNA non-specifically, leading to transcription initiation at random sites. This would produce the observed random initiation behavior.

Option 2: TATA-binding protein
TATA-binding protein is a eukaryotic transcription factor that binds to the TATA box in promoters. Bacteria do not use TBP in the same way, and the system described is bacterial. Its absence would not explain random initiation in a bacterial transcription unit, since TBP is not part of bacterial transcription machinery.

Option 3: rho factor
Rho factor is primarily involved in termination of transcription in bacteria, not initiation. A defect in rho would more likely affect termination efficiency or proceed to read-through issues, but would not directly cause initiation to occur at random sites. Therefore this does not account for random initiation.

Option 4: nucleoside triphosphates (NTPs)
NTPs are the substrates for RNA synthesis. If NTPs were missing or depleted, transcription would not proceed at all or would stall, rather than initiate at random positions. The presence of initiation implies NTPs are available, so this cannot explain random initiation.

Summary of reasoning: The hallmark of bacterial transcription initiation is promoter-specific binding by RNA polymerase holoenzyme, which requires the sigma factor to recognize promoters. Absence or malfunction of sigma would cause RNA polymerase to initiate transcription at various, non-promoter sites, yielding random initiation events. The other components either pertain to termination (rho), eukaryotic promoter recognition (TATA-binding protein), or substrate availability (NTPs) and do not align with the observed random initiation pattern.

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