1. Introduction to this semester’s Praktikum
Zasha Sommersemester 2019

2. The ribosome In E. coli rRNA Proteins Small subunit 16S 21
Large subunit
5S, 23S 31
Would be good to mention here that Iris is finding more r-leaders
Mention the blue things are various proteins.
Numbers of r-proteins are for E. coli

3. Ribosomal protein levels
Too low
Ribosome can‘t fold properly
Too high
Huge waste of energy
Ribosome might not fold properly

4. Ribosomal leaders regulate ribosomal protein levels

5. Alternate secondary structures and gene regulation
Illustration of regulation based on hiding SD sequence. SD=ribosome binding site.
From R2R paper, Fig. 6.
Drawing: (Weinberg & Breaker, 2011)

6. S8-binding r-leader resembles rRNA
S8 r-leader
rRNA (domain)
R-leader is beige, rRNA is blue
Figure: Iris Eckert and (Meyer, Microbiol Spectrum, 2018)

7. S8-binding r-leader resembles rRNA
S8 r-leader
rRNA (domain)
R-leader is beige, rRNA is blue
Figure: Iris Eckert and (Meyer, Microbiol Spectrum, 2018)

8. Interaction types r-leader imitate rRNA binding site
In primary / secondary structure
Only in 3-D structure
r-leader does not imitate rRNA
Example of does not mimic = E. coli S4 (according to Michell‘s review paper), although I think it was initially thought to mimic.

9. Results: L20 – known protein ligand rRNA binding site
Figure: Iris Eckert

10. Results: L20 – known protein ligand known r-leaders
Careful: there’s actually 2 E. coli r-leaders with distinct secondary structures that bind L20.
Figure: Iris Eckert

11. Challenge: Ligand is sometimes not clear
Example 1: Ligand must be S8
Example 2: Ligand could be S8, L5, L14, L6 or L18

12. Challenge: Binding site is often not clear
We want to know if the r-leader imitates the rRNA
Easier to analyze if we know which nucs. to look at.

13. Solution: wet-lab experiments
Expensive & time-consuming
(Sorry, I’m not that kind of biologist)

14. Solution: Covariation
Direct-Coupling Analysis
R-scape

15. Making the alignment

16. Making the alignment

17. Analyzing the rRNA/r-protein interaction
Find rRNA nucleotides nearby to the r-protein
Visualize that part of the rRNA

18. Analyzing the rRNA/r-protein interaction
View 3-D structure of ribosome (using PyMOL)
Find rRNA nucleotides nearby to amino acids in the r-protein (using PyMOL)
Map nucleotides onto rRNA alignment
Get rRNA alignment from Rfam Database
Add the rRNA sequence in the crystal structure (using Infernal)
Visualize that part of the rRNA
with R2R or YaaleRNA or your own script

19. Your goals Goal 1. Determine which gene encodes the ligand
Goal 2. Determine which nucleotides (in r-leader) & amino acids (in r-protein) interact
Goal 3. Compare the r-leader/r-protein interaction to the rRNA/r-protein interaction. Are they similar?

20. Plan With a validated r-leader (L10, L12)
Establish an RNA/protein multiple alignment
Find interactions with R-scape
Compare with rRNA using PyMOL and R2R
Then: everyone gets an unvalidated r-leader
Caveat: this is research. We don’t know if it will work.

21. Random tips Gaps can cause problems Some sequences will be truncated
(Because of incomplete genome sequences, part of the RNA or protein sequence is missing)
Alignments might be low quality
Maybe R-scape's E-value threshold (0.05) is too strict.
Fewer alignment columns  better signal
Interacting nucleotides / amino acids  high conservation
High conservation  little covariation

22. Data is not yet published
Please don’t give anyone outside of the class the data

23. Let’s look at the data…