RNA synthesis

Question 1

Describe the chemical reaction catalyzed by RNA polymerase and why it is unidirectional

Answer 1

RNA polymerase catalyzes a condensation rxn between 3’ OH on RNA chain and 5’ phosphate group. It is unidirectional in the 3’ direction because need 3’ OH.

Question 2

Distinguish five steps in the transcription cycle common to bacterial and eukaryotic RNA polymerases

Answer 2

initiation: 1) Polymerase binds promoter sequence of duplex DNA → closed complex 2) Polymerase melts duplex DNA forming transcription bubble → open complex 3) Polymerase catalyzes phosphodiester linkage of nucleotides elongation 5’ → 3’ polymerization of RNA strand. DNA-RNA hybrid region of about 9 newly synthesized BP Termination Transcription stops and polymerase dissociates at transcription stop site

Question 3

Name the four cellular RNA polymerases and their main functions

Answer 3

• Mitochondrial RNA polymerase: Transcribes mitochondrial genes
• RNA polymerase I: rRNA synthesis. Busiest ribosome in cell
• RNA polymerase II: mRNA, snRNA, miRNA, lncRNA
• RNA polymerase III: tRNA, 5S RNA, U6 snRNA, 7SK RNA

Question 4

Define a promoter and name sequence elements characteristic of promoters in human genes

Answer 4

• Region of DNA that acts to direct RNA polymerase to start transcription. Control direction and frequency of transcription.
• TATA box: TATAAA, important but not universal sequence.

Question 5

Describe how α‐amanitin and rifampicin block transcription

Answer 5

• Α‐amanitin: non-competitive inhibitor to RNA Pol II. Binds bridge helix and blocks RNA chain elongation.
• Rifampicin: was a common antibiotic, which binds bacterial RNA Pol and blocks the exit site, disallowing RNA chain elongation.

Question 6

Name four components of the RNA polymerase II pre‐initiation complex

Answer 6

1) TATA binding protein (TFIID)
2) TFIIA, B, E, F
3) TFIIH

Question 7

Describe the clinical syndromes caused by mutations in TFIIH subunits

Answer 7

mutations in XPD and XPB DNA helicase proteins and p44 lead to the following conditions:

• Xeroderma pigmentosum
• Cockayne’s syndrome
• Trichothiodystrophy

Question 8

Describe the three major ways in which most pre‐mRNA’s are processed

Answer 8

1) addition of a methylguanosine cap to the 5’ end. Methyl guanosine cap is added upside down (5’ to 5’) 2’ carbon methylated. Binds by attacking alpha phosphate on 5’ end. Cap protein (eIF4E oustide nucleus, CBC inside) recognizes and binds , protects from exonuclease activity, also upregulates translation
2) Introns are excised from RNA strand w/ use of spliceosomes. Recall lariat rxn mechanism
3) Poly A tail added to 3’ end (endonuclease slices strand after AAUAAA, opens up 3’OH group to which adenine is added)

Question 9

Compare and contrast a pre‐mRNA with a mature mRNA

Answer 9

pre-mRNA lacks several features of mature mRNA, namely:

1) pre-mRNA still contains introns
2) Lacks a 5-methylguanosine cap and associated eIF4E cap protein
3) Lacks poly A tail

Question 10

List the functions of the 5’ cap of the mRNA

Answer 10

• Stabilizes 5’ end against exonucleases
• CBC (cap binding complex in nucleus) important for future maturation steps (splicing, polyadenylation) Recognizes for export
• eIF4E, cap protein replaces CBC outside nucleus Removal eIF4E important for disposal

Question 11

List the three reactions required to add a 5’ cap to pre‐mRNA

Answer 11

1) triphosphatase lops off terminal gamma phosphate
2) guanylyltransferase adds GMP in reverse orientation
3) guanine 7 methyl transferase adds methyl group to seventh carbon position
4) second methylation rxn occurs at 2’ carbon
- This step is important in self vs. non-self recognition of RNA transcripts

Question 12

Recall the conserved sequences at the 5’ and 3’ ends of most introns and the consensus sequence at the polyA site

Answer 12

5’ end: GU 3’ end: AG closer to the 3’ end: A branch point PolyA site: AAUAAA

Question 13

Describe how alternative splicing permits multiple proteins to be produced by splicing defects

Answer 13

If splicing defects occur, then introns are incorrectly excised, meaning that once the RNA transcript is spliced back together, the reading frame for the ribosome has been changed. This change could result in additional or different amino acids being incorporated into the growing polypeptide chain during translation, therefore producing different proteins.

Question 14

Give examples of genetic disorders caused by splicing defects

Answer 14

• Abnormal splicing Cd44: contributes to tumor metastasis
• Spinal muscular atrophy (SMA) caused by mutation in SMN1 gene. Alternative SMN2 gene that is normally alternatively spliced (keeps exon 7 in nucleus, makes useless) drug therapy targets and turns off ISS (intronic splicing silencer) in exonic 7.

Question 15

Describe the function of U1 and U2 snRNA’s in splicing

Answer 15

2 of the 5 snRNA’s in spliceosome U1: binds to conserved 5’ sequence U2: binds to conserved A branch point U2AF: associates w/ U2 snRNA at 3’ Splice site These snRNA’s help spliceosome carry out two transesterification rxns

Question 16

Describe the two reactions that make the mature 3’ end of mRNA’s

Answer 16

1) endonuclease cleaves 3’ end just distal to AAUAAA sequence
2) polyadenylate polymerase adds adenines to exposed 3’ hydroxyl group

Question 17

Describe the relationship between 3’ end processing of the pre‐mRNA and termination of transcription at the end of a gene

Answer 17

Maturation 3’ end pre-mRNA is signal tells rna polymerase II to terminate when it reaches its termination sequence. If 3’ end doesn’t mature, transcription won’t terminate.

Question 18

Describe the two major functions of the mRNA’s poly A tail

Answer 18

1) stabilizes
2) enhances translation

Question 19

Give an example of how alternative poly A sites can be used to make more than one protein from a single gene Molecules

Answer 19

IGM Heavy chain gene:

Depending on which poly-A tail forms, the gene may either stay within membrane or be secreted (longer cut has hydrophobic region that sticks in membrane)