Lecture 5: RNA Binding Proteins

Question 1

How is RNA structured?

Answer 1

RNA has a ribose instead of a deoxyribose.
• It can form secondary structures.
• RNA helices. Internal loops. Anticodon loop is found in tRNA. GNRA loop is very stable.
• RNA can also form molecules with proteins, called ribonuclear proteins.
• RNA can also have catalytic activity.
• RNA is synthesised as a single strand. They most behave as such, but you can get double stranded RNA.
• We don’t see a B DNA like structure.
• RNA secondary structures fold in a predicted manner. The extra hydroxyl group affords flexibility and extra stability.
• You can get base stacking.
• Tertiary interactions are used in tRNA. There are some triple interactions and some Watson-Crick base pairs.

Question 2

How does the tRNA synthetase-tRNA complex work?

Answer 2

Synthetase recognises the correct tRNA.
• It’s like a protein-protein interaction.
• Folded molecules with a large interaction surface.
• Recognise the correct anticodon.
• Attach the amino acid at the acceptor CCA at the 3’ end.
• Sequence recognition is quite easy. Bases can be moved out.
• Not important to have sequence specific interactions. Its based on the fold and shape.
• Anticodon is splayed out and not stacked. This makes recognition easy.

Question 3

What are the different types of RNA-binding protein families?

Answer 3

• β sheets are often used as they match the flexibility.
• Arginine-rich motif. Found in many viruses. Example is phage lambda N-protein.
• All-helical proteins. Ribosomal L11.
• Zinc fingers. TFIIIA. Zinc fingers bind very differently

Question 4

How does the Rnt1p-RNA complex work?

Answer 4

• It has a dsRBM (double stranded RNA binding domain).
• Binds to RNA helices and cleaves them. It’s used in RNA silencing.
• Measures where the grooves are and how wide they are. This means it can bind to an A type helix. It doesn’t care about sequence.
• 3 protein regions contact RNA at 2’OH groups and phosphates.

Question 5

What is the Trp attenuator system?

Answer 5

It is a control system which uses the secondary structure of mRNA.
• Found in B. subtilis.
• Uses the TRAP protein. The TRAP protein binds.
• A and B can form a stem loop structure.
• C and D can also form a stem loop. But its smaller and not as stable.
• TRAP has sn type regions.
• TRAP destabilises the AB stem loop and causes the CD loop to form.
• RNA bases point inwards to the sn region binding pockets.
• There is 11-fold symmetry.
• L-trp is bound to each protein subunit.

Question 6

How does CRISPR work?

Answer 6

CRISPR-Cas9 is a genome editing technology.
• Cas9 is a protein. It is the nuclease.
• RNA is a guide (similar to RISC).
• Creates an RNA and DNA hybrid duplex.
• You just need guide RNA. Don’t need to worry about phage displays.

Question 7

How is the ribosome structured?

Answer 7

It is made up of a large and small subunit.
• The large subunit has a large RNA core with proteins stuck on the outside for stabilisation. Many duplexes, but also triples and tetraloops. Peptidyl transferase activity is catalysed by RNA.
• Small subunit decodes mRNA and checks pairing to tRNA anticodon. RNA is mainly in the core. Most of the RNA is in duplexes (50) but also triples and tetraloops. Shape is more irregular.
• Mechanisms were found from crystallography and EM high resolution structures.

Question 8

How does PAZ work?

Answer 8

• Binds to ssRNA.
• Creates small interfering RNAs.
• PAZ is part of a bigger protein. Its part of dicer.
• It measures length.
• Chopped up RNAs get recruited into the RISC complex.
• RISC binds to complementary mRNA and destroys it. siRNAs act as guide RNAs.
• Contacts to phosphate backbone. The 3’ end is recognised. It can be used as part of a system to measure length.