L16 : Gene Expression in Prokaryotes

Question 1

What are 3 important features of RNA?

Answer 1

• An abundance of alternative base pairs (only ~60 70% bps are WC in structured RNA)
• Multiple gaps and loops
• Highly structured

Question 2

How do single nt changes affect RNA?

Answer 2

Can drastically alter conformation and disrupt function due to RNA structural sensitivity

Question 3

What are the 4 types of RNA interactions?

Answer 3

• RNA-RNA (intra-inter)
• RNA-DNA
• RNA-protein (regulators, chaperones, targets)
• RNA-metabolite (riboswitches)

Question 4

What are the main categories of riboregulators in bacteria?

Answer 4

Cis acting
- RNA leaders
- RNA thermometers
- riboswitches

Trans acting
- sRNAs (cis or trans encoded)
- dual function sRNAs

Question 5

What defines a ncRNA?

Answer 5

Transcript that does not encode protein or peptide
May or may not have regulatory function

Question 6

What is the difference between cis and trans acting RNAs?

Answer 6

Cis - regulate the same transcript they are part of (eg. leaders, 3’UTRs)

Trans - act on separate transcripts (eg. sRNAs, antisense RNAs)

Question 7

What are dual function sRNAs?

Answer 7

Both regulate RNA and encode small protein

Question 8

List some RNA-related NGS methods?

Answer 8

RNA-seq: expression & transcript structure

dRNA-seq: maps TSS via triphosphate enrichment

Term-seq: maps 3’ ends

Ribo-seq: identifies translated mRNA regions

Grad-seq: RNA-protein interactions

Dual RNA-seq: host-pathogen interactions

Single-cell RNA-seq: cell-cell variation

RIL-seq: RNA-RNA interaction mapping via Hfq

Question 9

What is Illumina used extensively for?

Answer 9

• Differential gene expression analysis
• TSS mapping
• Term-seq for mapping of RNA 3’ ends

Question 10

What are limitations of Illumina?

Answer 10

Relatively short reads
- Can lead to misassembly or repeat sequences

Reliance on PCR amplification for library prep
- Can introduce coverage bias
- GC poor and rich sequences may be underrepresented

Question 11

What is the principle of PacBio (SMRT) sequencing?

Answer 11

Measures incorporation and release of fluorescently labelled dNTPs by DNAP in real time.

Question 12

What is the principle of Nanopore sequencing?

Answer 12

Identification of bases through measuring characteristic disruptions in ionic current as single nucleotides pass through nanapore

Can also identify base modifications

Question 13

What are the main components of a bacterial operon?

Answer 13

5’ UTR
TSS
Internal genes
TTS
3’ UTR
- Internal TSS or TTS

Question 14

Explain how operon expression is characterised with RNA-seq?

Answer 14

1. Isolate and fragment RNA
2. Prepare library and sequence
3. Map reads to reference genome

Allows quantification of gene expression and discovery of novel transcripts

Question 15

What is dRNA-seq used for?

Answer 15

To map transcriptional start sites