Transcription in prokaryotes

• Transcription in Bacteria can be conveniently divided into three stages:

Initiation, 

• Initiation of transcription begins at the promoter, the region where the transcription enzyme RNA polymerase binds the DNA. 
• The promoters are usually located on the upstream of the genes to be transcribed. 
• The promoters of the prokaryotes differ in their sequences, though there are two conserved sequences observed. 
• These two sequences are located approximately 10 and 35 base pairs upstream of the transcription start site.
• The transcription start point, which the first base transcribed is termed the +1 site or the initiation site. 
• The region on the upstream of this initiation site is denoted by a negative (-) sign and the ones on the downstream are denoted by a positive (+) sign.

• Hence the conserved upstream sites are referred to as -10 and -35 regions. 
• The -10 region has the conserved sequence of TATAAT and the -35 region has TTGACA sequence 
• The σ subunit of RNA polymerase (RNA pol) binds specifically to both, the -35 and -10 regions, and loads the RNA pol onto the promoter. 
• The binding of the RNA pol to the promoter results in formation of a closed-promoter complex.
• The RNA pol then interacts with the DNA and unwinds around a 15 bases long region at the initiation site to create a transcription bubble, collectively called open-promoter complex. 
• As the initiation site unwinds, single-stranded DNA is exposed and can be used as a template for transcription  .  
• The strand which is transcribed is known as the template strand or antisense strand. 
• The other strand is called as coding strand or sense strand
• RNA pol initially initiates abortive transcription, wherein short cycles of synthesis and release of short RNA transcripts of approximately 10 nucleotides take place till RNA polymerase-promoter initial transcribing complex leaves the promoter. 
• The RNA pol escapes the promoter and initiates productive synthesis of RNA, forming RNA polymerase-DNA elongation complex

2. Elongation:

• RNA polymerase-DNA elongation complex is stable and processive. 
• It carries out transcription on an average rate of 30 – 100 nucleotide/sec.
• RNA polymerase is the principle protein, which adds the nucleotides. 
• In most prokaryotes, a single type of RNA pol transcribe all types of RNA. 
• The core RNA pol is made up of 5 subunits, which are conserved in prokaryotes as well as eukaryotes. 
• Prokaryotic core enzyme consists of two copies of α, one copy of each- β, β’, and ω subunits. 
• The core enzyme can bind the template DNA and synthesize RNA.
• σ factor binds the core enzyme to form the holoenzyme and helps in recognising the promoter.
• α subunit interacts with various transcription factors and helps regulate transcription. 
• It also has DNA binding site with help of which it binds upstream promoter DNA. 
• The RNA pol has two α subunits forming a homodimer which binds β and β’ subunits.
• β and β’ subunits are the largest subunits which form the catalytic center of RNA synthesis and have binding sites for double-stranded downstream DNA, DNA/RNA hybrid and RNA.
• The ω subunit has been linked recently with RNA pol stability and specificity
• σ factor associates with the core enzyme for promoter recognition and it dissociates from the core enzyme once RNA pol starts processive RNA synthesis.
• RNA pol catalyzes the polymerization of ribonucleoside 5′-triphosphates (NTPs) using the DNA as template. 
• The ribonucleoside triphosphates are added to the 3′ end, i.e. mRNA synthesis takes place in the 5′ to 3′ direction. 
• This extension of RNA chain occurs de novo and doesn’t need a primer.
• In prokaryotes, the genomic DNA usually contain no introns and the transcript synthesized may contain information for more than one protein. 
• Such transcripts are called as polycistronic mRNA.

3. Termination:

• The transcription has to be terminated to release the newly synthesized mRNA. 
• The termination is brought about by two different mechanism, one is rho independent or RNA-based and the other is rho dependent or protein-based.

Rho independent or RNA based:

• RNA based termination employs intrinsic (in RNA structure itself) terminators having two structural features, GC-rich inverted repeat with several intervening nucleotides, followed by a stretch of U residues in the transcribed RNA.
• When the GC-rich inverted repeats are transcribed, the RNA regions with self complementary sequences base-pair with one another and form a stem-loop structure . 
• This stem-loop structure interacts with the RNA polymerase and causes it to pause.
• The following stretch of U residues at the 3′ end of the transcript, which are weakly base-paired with the A residues of the template DNA, create a very unstable region.
• The stem loop followed by the weakly bond stretch together leads to the release of the RNA transcript from the transcription complex.

Rho dependent or protein-based:

• This mechanism was first recognized in λ-phage DNA. 
• It involves a protein called Rho factor, which is a hexameric protein. 
• Rho is a RNA/DNA helicase or translocase, that causes dissociation of RNA pol from DNA template and releases RNA. It also has ATPase region.
• In rho dependent termination, there are specific sites, transcribed called the rho–dependent terminators. 
• These contain two separate sites; a rho utilization site called the ‘rut site’ and a downstream transcription stop point also called as ‘tsp site’
• Rho binds RNA at the transcribed ‘rut site’ region. 

• This binding activates RNA-dependent ATPase activity of Rho. 
• Rho moves along the RNA till it reached the RNA pol. 
• The energy derived from the hydrolysis of ATP is used by the rho, with helicase activity, to unwind RNA-DNA hybrid from RNA pol and results into termination
• In prokaryotes, protein synthesis or the translation starts simultaneously with the transcription, while the mRNA is still being synthesized. 
• This is possible due to the lack of nucleus or any type of compartmentalisation. 
• Hence, the transcript is immediately translated to synthesize the corresponding protein.

Initiation Stage

1. The -10 region in prokaryotic promoters is also called:
   A. TATA box
   B. Pribnow box
   C. CAAT box
   D. GC box
   Answer: B
2. The conserved sequence of the -35 region in prokaryotic promoters is:
   A. TATAAT
   B. TTGACA
   C. AATTGG
   D. CCAAT
   Answer: B
3. The σ subunit of RNA polymerase is essential for:
   A. DNA unwinding
   B. Promoter recognition
   C. RNA elongation
   D. Termination
   Answer: B
4. The transcription start site is designated as:
   A. -1 site
   B. +1 site
   C. 0 site
   D. Initiation codon
   Answer: B
5. The closed-promoter complex becomes an open-promoter complex after:
   A. DNA unwinding
   B. Sigma factor binding
   C. Rho factor binding
   D. RNA primer synthesis
   Answer: A

RNA Polymerase Structure

6. The core enzyme of prokaryotic RNA polymerase includes all subunits EXCEPT:
   A. α
   B. β
   C. σ
   D. β’
   Answer: C
7. Which subunit of RNA polymerase binds upstream promoter DNA?
   A. α
   B. β
   C. σ
   D. ω
   Answer: A
8. The catalytic site for RNA synthesis is primarily located in the:
   A. β subunit
   B. β’ subunit
   C. α subunit
   D. σ subunit
   Answer: A
9. The ω subunit is associated with:
   A. Promoter recognition
   B. RNA pol stability
   C. DNA unwinding
   D. Termination
   Answer: B
10. The holoenzyme is formed by the core enzyme plus:
    A. Rho factor
    B. σ factor
    C. tRNA
    D. Helicase
    Answer: B

Elongation Stage

11. RNA synthesis in prokaryotes occurs in the:
    A. 3’→5’ direction
    B. 5’→3’ direction
    C. Both directions
    D. Random direction
    Answer: B
12. Abortive transcription refers to:
    A. Synthesis of short RNA fragments
    B. Premature termination
    C. Rho-dependent termination
    D. DNA repair
    Answer: A
13. Polycistronic mRNA contains information for:
    A. One protein
    B. Multiple proteins
    C. rRNA only
    D. tRNA only
    Answer: B
14. During elongation, the transcription bubble moves:
    A. Upstream
    B. Downstream
    C. Bidirectionally
    D. Stays fixed
    Answer: B
15. RNA polymerase does NOT require:
    A. DNA template
    B. Ribonucleotides
    C. Primer
    D. Mg²⁺ ions
    Answer: C

Termination Stage

16. Rho-independent termination relies on:
    A. Stem-loop structures and U-rich regions
    B. ATP hydrolysis
    C. Sigma factors
    D. DNA ligase
    Answer: A
17. The stem-loop structure in RNA causes RNA polymerase to:
    A. Initiate transcription
    B. Pause
    C. Bind sigma factor
    D. Repair DNA
    Answer: B
18. Rho factor is a:
    A. DNA helicase
    B. RNA/DNA helicase
    C. Sigma factor
    D. Core enzyme subunit
    Answer: B
19. The “rut site” is essential for:
    A. Rho-dependent termination
    B. Promoter recognition
    C. RNA splicing
    D. DNA replication
    Answer: A
20. In Rho-dependent termination, Rho binds to:
    A. DNA
    B. RNA
    C. Sigma factor
    D. RNA polymerase
    Answer: B

General Mechanisms

21. Transcription and translation occur simultaneously in prokaryotes due to:
    A. Absence of nucleus
    B. Presence of introns
    C. Polycistronic mRNA
    D. Sigma factors
    Answer: A
22. The template strand is also called:
    A. Coding strand
    B. Sense strand
    C. Antisense strand
    D. Leading strand
    Answer: C
23. Which statement is FALSE about prokaryotic transcription?
    A. Requires primers
    B. Uses σ factors
    C. Synthesizes polycistronic RNA
    D. Occurs in the cytoplasm
    Answer: A
24. The RNA-DNA hybrid during elongation is typically:
    A. 8–9 bp long
    B. 15–20 bp long
    C. 50 bp long
    D. 100 bp long
    Answer: A
25. Prokaryotic RNA polymerase can:
    A. Proofread RNA
    B. Unwind DNA without helicase
    C. Synthesize DNA
    D. Add primers
    Answer: B

Advanced Concepts

26. The “tsp site” in Rho-dependent termination refers to:
    A. Transcription start point
    B. Transcription stop point
    C. Template strand promoter
    D. Terminator sigma protein
    Answer: B
27. Rho utilizes energy from:
    A. GTP hydrolysis
    B. ATP hydrolysis
    C. DNA supercoiling
    D. RNA splicing
    Answer: B
28. In Rho-independent termination, the weak bonding region consists of:
    A. A-U base pairs
    B. G-C base pairs
    C. T-A base pairs
    D. C-G base pairs
    Answer: A
29. Which subunit dissociates after initiation?
    A. σ factor
    B. β subunit
    C. α subunit
    D. ω subunit
    Answer: A
30. Abortive transcription ensures:
    A. Correct promoter escape
    B. DNA repair
    C. Termination
    D. Primer synthesis
    Answer: A

Miscellaneous

31. The coding strand has the same sequence as the:
    A. Template strand
    B. Newly synthesized RNA
    C. Antisense strand
    D. rRNA
    Answer: B
32. Transcription in prokaryotes is inhibited by:
    A. Rifampicin
    B. Actinomycin D
    C. α-amanitin
    D. Tetracycline
    Answer: A
33. The average rate of RNA elongation in prokaryotes is:
    A. 10–20 nucleotides/sec
    B. 30–100 nucleotides/sec
    C. 200–300 nucleotides/sec
    D. 500 nucleotides/sec
    Answer: B
34. Which is NOT a function of the σ factor?
    A. Promoter recognition
    B. DNA unwinding
    C. Core enzyme binding
    D. Termination
    Answer: D
35. The ω subunit is found in:
    A. Core enzyme
    B. Holoenzyme
    C. Rho factor
    D. Ribosomes
    Answer: A

True/False Style

36. Which statement is TRUE?
    A. Prokaryotic mRNA has introns.
    B. RNA pol requires a primer.
    C. Sigma factors are part of the core enzyme.
    D. Rho binds RNA during termination.
    Answer: D
37. Which is NOT a feature of Rho-independent termination?
    A. Stem-loop structure
    B. U-rich region
    C. Rho helicase activity
    D. Weak RNA-DNA binding
    Answer: C
38. The β’ subunit binds:
    A. DNA template
    B. Sigma factor
    C. RNA primer
    D. Ribosomes
    Answer: A
39. Transcription and translation coupling is possible because:
    A. mRNA is processed in the nucleus
    B. No nuclear membrane exists
    C. DNA is circular
    D. RNA pol is fast
    Answer: B
40. The σ factor is a:
    A. Transcription factor
    B. Translation factor
    C. Replication enzyme
    D. Splicing factor
    Answer: A

Scenario-Based

41. If the -35 region is mutated, what process is affected?
    A. Initiation
    B. Elongation
    C. Termination
    D. Translation
    Answer: A
42. A stem-loop followed by a poly-U region in RNA signals:
    A. Initiation
    B. Rho-dependent termination
    C. Rho-independent termination
    D. Translation
    Answer: C
43. Rho factor deficiency would primarily disrupt:
    A. Promoter escape
    B. Protein-based termination
    C. RNA-based termination
    D. DNA replication
    Answer: B
44. A mutation in the β subunit would likely affect:
    A. Promoter recognition
    B. Catalytic RNA synthesis
    C. Sigma factor binding
    D. DNA unwinding
    Answer: B
45. Absence of σ factor would prevent:
    A. Core enzyme assembly
    B. Promoter binding
    C. RNA elongation
    D. Termination
    Answer: B

Final Questions

46. Which enzyme is responsible for prokaryotic transcription?
    A. DNA polymerase III
    B. RNA polymerase
    C. Reverse transcriptase
    D. Ligase
    Answer: B
47. The “open-promoter complex” refers to:
    A. Unwound DNA at the start site
    B. Sigma factor binding
    C. Rho factor activity
    D. Terminator sequence
    Answer: A
48. In prokaryotes, RNA polymerase binds to:
    A. Ribosomes
    B. Promoter
    C. Terminator
    D. tRNA
    Answer: B
49. Which process occurs first during transcription?
    A. Elongation
    B. Termination
    C. Initiation
    D. Splicing
    Answer: C
50. The primary function of Rho helicase is to:
    A. Unwind RNA-DNA hybrids
    B. Synthesize RNA
    C. Bind promoters
    D. Proofread RNA
    Answer: A