9. Products of transcription: mRNA

Question 1

Whats does mRNA start as in the nucleus ?

Answer 1

hnRNA

Question 2

heterogeneous nuclear RNA (hnRNA)

Answer 2

• in nucleus
• heterogenous in size and sequence
• produced by RNA polymerase II; a copy of the gene
• has intervening non-coding regions-introns- which are spliced out, forming the smaller cytoplasmic form (exons are parts retained)
• the half-life of new hnRNAs is only a few minutes as they get processed into mRNA (as it is made, hnRNA is covered by small RNAs and proteins)

Question 3

Pulse chase experiment:
experiment: processing of nuclear RNAs
expose cells to [32P]-labelled nucleotides for 30 min

Answer 3

• they purified nuclear RNA and separated according to size
• measure radioactivity in each size group
• large RNA molecules take up the label (80s, 50,000 bases)
• chase with unable phosphate for 3 hours- labeled fraction is much smaller now

Question 4

pulse chase experiment conclustion

Answer 4

the primary transcript is made processed and shortened during that time

Question 5

RNA splicing

Answer 5

-hnRNA, primary transcript
*made by RNA polymerase II
*doesnt last long, half life of 15 min, processed as it is made
-exon-contains the final coding sequence
-intron-will be spliced out
refer to slide 7 for mature, cytoplasmic mRNA pic

Question 6

mRNA Splicing page 9

Answer 6

• introns much be removed with precision: one base off and the protein is wrong
• there are highly conserved, recognizable sequence regions called the splice-sites
  
  • consensus sequence:same for almost all genes
• splicing is done by a number of protein/RNA molecules

Question 7

mRNA splicing proteins/RNA molecules

Answer 7

• small nuclear RNAs (snRNAs) bind to proteins (the U Proteins) to form snRNPs- “snurps”
• the RNAs recognized the splice sites by base pairing
• the complex of snRNA, hnRNA and proteins is called a spliceosome
• spliceosome is formed in nucleus, as transcription takes place

Question 8

spliceosome is comprised of :

Answer 8

1. snRNPs which are snRNAs bound to specific proteins (the U proteins)
2. other proteins that hold the assembly together

Question 9

U proteins

Answer 9

recognize each other and bind to their substrates by base-pairing using their snRNAs

Question 10

spliceosome u proteins accomplish the following

Answer 10

matching
cutting
ligation of the hnRNA

Question 11

Intron splicing process

Answer 11

1. hnRNA is substrate
2. U1 snRNA binds to the 5’ side of the splice site by base pairing snRNA to substrate
3. U2 snRNA binds to certain region of intron, the branch point
4. the U4/U6 and U5 bind displacing the u1(splicosome is assembling a loop) U4 is displaced, U6 switches to bind the U2
5. Now U6 acts as an enzyme (cuts 5’ splice site, cuts 3’ splice site, simultaneously joins the cut exon ends together , releases the intron)
6. during the cutting process U5 has been holding the two ends

Question 12

ribozymes -catalytic RNA

Answer 12

• in some eukaryotes (protozoans) as well as some mitochondria and chloroplasts, the splicing reaction is accomplished by RNA only(no protein)
• RNA is acting as a catalyst
• this ribozyme is actually a part of the intron
• it is likely that the catalytic function of the snRNPs is actually in the RNA portion (snRNAs)

Question 13

messenger RNA structure

Answer 13

1. 5’ cap of methylguanosine
2. a 5’ untranslated region (5’UTR)
3. a continuous sequence of nucleotides encoding a protein
4. an untranslated region 3’ to the coding region (3’UTR)
5. a tail of about 250 “A” nucleotides =poly A-tail