15. Rho factor is involved in termination of transcription in prokaryotes. Genetic manipulations indicate that Rho-dependent termination requires the presence of a specific recognition sequence on the newly synthesized RNA upstream of the termination site. The recognition sequence must be on the necent RNA rather then the DNA, as demonstrated by Rho’s inability to terminate transcription in the presence of pancreatic RNAse.  The essential features of this termination site have not been fully elucidated. Construction of synthetic termination sites indicates that it consists of 80 to 100 nts that lack a stable secondary structure and contain multiple regions that are rich in C and poor in G. Which of the following is/ are suggested by the above observation?
(1) Rho factor attaches to nascent RNA at its recognition sequence and then migrates along the RNA in the 5′–>3^. direction until it encounters an RNAP paused at the termination site and disrupts the transcriptional complex by acting as an RNA-DNA helicase.
(2) Rho unwinds the RNA-DNA duplex forming the transcription bubble, thereby releasing the RNA transcript.
(3) Rho factor attaches to the RNA at its recognition sequence while RNA is in the RNA-DNA hybrid condition.
(4) There may be other factors and hence Rho factor does not need to unwind the RNA-DNA hybrid to release the transcript.

Detailed Explanation of Rho-Dependent Termination

The Rho factor is a hexameric ATP-dependent RNA helicase found in prokaryotes (like E. coli) that terminates transcription at specific sites in a Rho-dependent manner.

Based on the Question Passage:

• Rho binds to nascent RNA (not DNA) at a specific recognition sequence called the Rho utilization site (rut site).

• Rut sites are:

  • C-rich, G-poor

  • Unstructured (lack stable secondary structures)

  • Located ~80–100 nt upstream of the termination site

• Rho fails to function if RNA is digested by pancreatic RNase, confirming that it acts on free RNA, not DNA.

• The mechanism involves chasing the paused RNA polymerase and disrupting the RNA-DNA hybrid.

 Statement-wise Evaluation:

(1) Rho factor attaches to nascent RNA at its recognition sequence and then migrates along the RNA in the 5′ → 3′ direction until it encounters an RNAP paused at the termination site and disrupts the transcriptional complex by acting as an RNA-DNA helicase.

• True / Correct

• Rho travels 5′ → 3′ using ATP hydrolysis.

• When it catches up with a paused RNA polymerase, it uses its helicase activity to unwind the RNA-DNA hybrid, causing termination.

• This is the core model of Rho-dependent termination.

(2) Rho unwinds the RNA-DNA duplex forming the transcription bubble, thereby releasing the RNA transcript.

• Incorrect

(3) Rho factor attaches to the RNA at its recognition sequence while RNA is in the RNA-DNA hybrid condition.

• False

• Rho binds to free, single-stranded RNA, not RNA that’s still base-paired with DNA (RNA-DNA hybrid).

• The rut site must be accessible, not hybridized.

(4) There may be other factors and hence Rho factor does not need to unwind the RNA-DNA hybrid to release the transcript.

• False

• Rho must unwind the RNA-DNA duplex to terminate transcription.

• Other factors might assist in some contexts, but unwinding by Rho is essential in Rho-dependent termination.