I. DNA & RNA | 11B. Termination of transcription in prokaryotes; post-transcriptional RNA modifications in prokaryotic cells

Question 1

What is a transcription terminator?

Answer 1

a section of nucleic acid sequence that marks the end of a gene/operon in genomic DNA during transcription.

Question 2

The role of the transcription terminator

Answer 2

This sequence mediates transcriptional termination, by providing signals in the newly synthesized mRNA, that trigger processes which release the mRNA from the transcriptional complex.

Question 3

Termination in prokaryotes is distinguished into 2 classes which are…

Answer 3

1. (rho) ρ-independent
2. (rho) ρ-dependent
   (protein factor = ρ)

Question 4

2 main features of ρ-independent termination
(transcription terminators require…; The hairpin formation…)

Answer 4

• transcription terminators require the formation of a GC rich hairpin structure on the elongating transcript, which results in the disruption of the mRNA-DNA-RNA polymerase ternary complex
• The hairpin formation causes RNA polymerase destabilization, leading to a dissociation of the complex

Question 5

What are the 4 steps of ρ-independent termination?

Answer 5

1. A region produces an RNA transcript with self-
   complementary sequences, permitting the formation of a hairpin structure centered 15 to 20 nucleotides before the projected end of the RNA strand
2. RNA polymerase transcribes ‘’A = adenine’’ rich sequence on DNA
   -> ‘’U = uracil’’ rich sequence created on RNA
3. The hairpin makes RNA polymerase pause after having the adenines transcribed
4. The growing hairpin will then pull the RNA strand out of the loose (A:U) DNA-RNA hybrid helix
   => terminate transcription

Question 6

3 main features of ρ-dependent termination

Answer 6

• The ρ-dependent terminators require a protein called Rho-factor (ρ), which has RNA helicase activity to disrupt the mRNA-DNA-RNA polymerase transcriptional complex
• Terminators lack the sequence of repeated A residues in the template strand, but usually include a CA-rich sequence called a rut (ρ utilization) element
• The rut serves as an mRNA loading site and as an activator for Rho -> Activated Rho will hydrolyze ATP and translocate down the mRNA

Question 7

3 main steps of ρ-dependent termination

Answer 7

1. The helicase Rho-factor follows the RNA polymerase
2. A hairpin makes the polymerase stops, so Rho-factor catches up
3. The Rho-factor then creates a loop of polypeptide chain that stops elongation by separating the DNA-RNA double helix

Question 8

3 main steps of ρ-dependent termination

Answer 8

1. The helicase Rho-factor follows the RNA polymerase
2. A hairpin makes the polymerase stops, so Rho-factor catches up
3. The Rho-factor then creates a loop of polypeptide chain that
   stops elongation by separating the DNA-RNA double helix

Question 9

Give a Summary of prokaryote transcription:

Answer 9

• Step 1: The RNA polymerase holoenzyme (polymerase core enzyme + σ-factor) assembles and then locates a promoter DNA sequence to begin
• Step 2: The polymerase opens (unwinds) the DNA at the position at which transcription is to, and begins transcribing
• Step 3: This initial RNA synthesis (abortive initiation) is relatively inefficient as short, unproductive transcripts are often released
• Step 4: Once RNA polymerase has managed to synthesize about 10 nucleotides of RNA, it breaks its interactions with the promoter DNA
• Step 5: Eventually, the σ-factor releases as the polymerase tightens around the DNA and shifts to the elongation mode of RNA synthesis, moving along the DNA
• Step 6 + 7: During the elongation mode, transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal
• Step 8: termination signals are typically encoded in DNA and many function by forming an RNA hairpin-like structure that destabilizes the polymerase’s hold on the RNA
  ****In bacteria, all RNA molecules are synthesized by a single type of RNA polymerase

Question 10

What does the primary transcript contain?

Answer 10

Ribosomal RNAs and some transfer RNAs in a continuous strand

Question 11

What happen during Processing of prokaryotic rRNA and tRNA?

Answer 11

Both genes for rRNA and tRNA are clustered in the nucleolus, thus the nucleolus can be thought of as a large factory where noncoding RNAs are being transcribed.

(All rRNA genes have an important role in forming the nucleolus.)

After synthesis of mRNA (transcription), the mRNA are ready to start translation. Since translations require both rRNA (to make the ribosomal subunits) and tRNA (make protein), they need to be produced

Question 12

3 steps of maturation of tRNAs

Answer 12

1/ Primary RNA is chopped by various endonuclease (including RNase P)
2/ RNase D exonuclease trims the 3’ end
3/ tRNA nucleotidyltransferase extends the new 3’ end with CCA
-> The process is continued by nucleotide/base modifications (e.g, hypoxanthine, thymine)