L8 RNA and gene expression

Question 1

RNA structure

Answer 1

Difference to DNA:
Deoxyribose sugar backbone (OH) hydroxyl group so it gets degraded rapidly
T nucleotide is replaced with Uracil (U)

Question 2

RNA structures

Answer 2

Intra base pairing events, hence:

Stem loop and folding occurs, can contain double helical structures.

Question 3

Types of RNA

Answer 3

mRNA(messenger) > translation > protein (500-1000bp)
tRNA(translation)
rRNA (ribosomes)

Question 4

Transcription basic concepts

Answer 4

DNA RNA > Protein
The amount of RNA:
* Indicates gene expression levels
* Higher abundance of mRNA may result ni higher abundance of protein.

-Detection and quantification of mRNA abundance allows studies of gene expression and gene regulation-

Question 5

Transcription process (prokaryotic)

Answer 5

Need :
Promoter - provides signal for RNA polymerase to bind.
mRNA is synthesized by RNA polymerase in 5’ to 3’
Until transcription terminator structure is found

NOTE: The transcribed region is longer relative to translated region.

Question 6

Transcriptional fusions

Answer 6

Uses a reporter gene under the control of promoter
It gives an indirect estimation of gene expression
Test gene expression
Convenient in-situ gene expression

Question 7

Transcriptional fusion process

Answer 7

Clone promoter
Use promoter probe - a promoter-less gene (reporter gene)
Hence its fusion of promoter to a reporter gene

ACTIVITY OF A REPORTER PROTEIN IS PROPORTIONAL TO EXPRESSION OF CLONED PROMOTER.

Question 8

Transcriptional fusion: application

Answer 8

Alk5 is a receptor kinase involved in vascular endothelium development. Fused to lacZ in mouse embryo.

Streptomyces coelicolor “dpsA” gene.
dpsA is fused to mCherry (auto florescent)
Can see where the gene is expressed within the bacteria

Question 9

Analyzing RNA disadvantages

Answer 9

disadvantages:
indirect
reporter gene remain active long after gene expression has stopped
only strongly expressed promoters are detected

Question 10

RNA isolation

Answer 10

Isolated from cells
isolation is more difficult than DNA
RNases in the environment and within cells will degrade isolated RNA
all equipment must be treated so ribonucleases are destroyed
DNA must be completely removed from RNA sample by endonuclease treatment [DNases]

Question 11

Separation of RNA

Answer 11

Agarose electrophoresis
RNA sugar phosphate backbone has -ve charge
RNA must be heated before eletrophoresis to disrupt secondary structures. Formaldehyde is added to ensure denaturing.

MUST CONTROL QUALITY OF RNA SAMPLE

Question 12

Northern blot

Answer 12

Used to detect RNA
separate RNA sample by electrophoresis
transfered to a membrane
RNA is crosslinked to nylon membrane using UV
Hybridise membrane with DNA probe (florescent)
Probe must be antisense of target for it to hybridise with mRNA

RNases must not be present

Question 13

Nuclease protection assay

Answer 13

Based on combining DNA and RNA
sensitive method
more efficient, does not require membrane binding
detect little as 4000 copies
use map transcription start sites.

PCR
Use a radioactive group to 1 of the strand
Denature radioactive strand
Mix DNA with RNA, to get hybrid. both single strands have single strand overhangs.
Degrade overhand with s1 nuclease - degrades single strand, but not double strand.
Gel electrophoresis used to detect.

Question 14

In-situ RNA hybridisation

Answer 14

Detection os transcripts in a tissue,cell.

Use fluorescent markers and hybridise to respective DNA gene.