Lec 47: RNA Synthesis and Processing

Question 1

Define Transcription

Answer 1

Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase.

Question 2

Name the 4 main functions of RNA Polymerase

Answer 2

1) Search for and bind promoters- aided by other proteins
2) Unwind a short stretch of DNA
3) Initiate and catalyze polymerization reaction that yields RNA
4) Detect termination signals and halt reaction

Question 3

Describe the molecular activity of RNA Polymerase

Answer 3

• RNA polymerase acts by adding nucleotides to the growing 3’ end using the template strand of DNA (adds U instead of T).
• Occurs by nucleophilic attack by the 3’ hydroxyl on alpha phosphate that generates new 3’ to 5’ bond, releasing pyrophosphate which is then hydrolyzed.
• RNA polymerase catalyzes the formation of ester bonds between nucleotides that base-pair with the complementary nucleotides on the DNA template.

Question 4

Compare/Contrast DNA Polymerase and RNA Polymerase

Answer 4

• RNA polymerase acts very similarly to DNA polymerase in that it polymerizes along one strand of DNA from the 5’ to 3’ direction using the opposite strand as a template.
• However, the job of RNA polymerase is to transcribe certain portions of DNA (as opposed to the whole chromosome by DNA polymerase).
• In contrast to DNA polymerase, RNA polymerase does not require a primer.
• Also in contrast to DNA polymerase, RNA polymerases lack 3’ to 5’ exonuclease activity, although they do perform rudimentary error checking through a different mechanism.

Question 5

List the specificities of the eukaryotic RNA polymerases

Answer 5

Eukaryotes have three RNA polymerases
RNA Polymerase I: produces rRNA
RNA Polymerase II: produces mRNA and micro RNA (miRNA)
RNA Polymerase III: Produces tRNA and other small RNAs
All three RNA polymerases have the same mechanism of action however they recognize different types of promoters.

(Remember prokaryotes only have one RNA Polymerase)

Question 6

Describe the makeup of Prokaryotic RNA polymerase

Answer 6

Prokaryotic RNA polymerase has 5 subunits: two alpha subunits, a beta subunit, and beta prime subunit, and a sigma subunit. The core enzyme is composed of the alpha and beta subunits. During initiation, the sigma subunit joins.

Question 7

What are the two sequences in DNA are recognized by RNA polymerase as indicating a start of transcription?

Answer 7

1) The sequence between -7 and -10 called the -10 region (TATAAT)
2) The sequence at -35 (TTGACA)

Question 8

Describe Prokaryotic Transcription Termination

Answer 8

• RNA polymerase pauses at a U-U-U-U sequence following a hairpin
• Rho protein (RNA- dependant ATPase, acts like a motor) travels along mRNA, catches the polymerase from behind, pulls the RNA out, and terminates transcription.

Question 9

Differentiate between eukaryotic TATA box and prokaryotic TATAAT sequence

Answer 9

Despite the similar sequence between eukaryotic TATA box and prokaryotic TATAAT sequence, they are not functionally related. TATA box binds TATA box protein (TBP) that recruits RNA polymerase. TATAAT binds RNA polymerase directly. In addition, TATAAT region in prokaryotes must be exactly -10 from start site while TATA box in eukaryotes can be anywhere in region.

Question 10

Describe RNA polymerase activity in eukaryotes

Answer 10

-Initial binding of the TATA box binding protein (TBP) occurs.
-Next, coactivators bind with or right after TBP.
(Note: there may not always be a TATA box but TBP are usually always required. In these instances, coactivators bind first to recruit TBP to binding site.)
-Once these proteins have bound, binding of TFII A and TFII B to DNA occurs.
-This is followed by binding of TFII F which recruits RNA polymerase to the promoter site.
-Next, TFII E and TFII H (TFII H= helicase, unwinds DNA and phosphorylates RNA polymerase) bind.
-Transcription occurs.

Question 11

Describe Eukaryotic Transcription Termination

Answer 11

• Main signal for RNA polymerase to terminate transcription is a polyadenylation signal.
• Almost all mRNA in eukaryotes have a polyA tail added to RNA that is encoded in the DNA (about 150-250 residues).
• This tail is not added by RNA polymerase. RNA polymerase encounters polyadenylation signal, nuclease cleaves mRNA, terminates transcription. At the cleavage site, poly(A) polymerase adds many A resides.

Question 12

Name the steps of pre-mRNA is modification in prokaryotes and eukaryotes

Answer 12

Prokaryotes: mRNAs are not processed.
Eukaryotes: pre-mRNAs are transcribed separately in the nucleus. 3 essential processing: 5’ cap, Poly A tail, intron splicing

Question 13

Describe rRNA production in prokaryotes

Answer 13

• rRNA is produced as a large transcript that is cleaved, producing the 16S rRNA that appears in the 30S ribosomal subunit and the 23S and 5S rRNAs that appear in the 50S ribosomal subunit.
• The 30S and 50S ribosomal subunits combine to form the 70S ribosome.

Question 14

Describe rRNA synthesis in eukaryotes

Answer 14

A 45S precursor is produced by RNA polymerase I from rRNA genes located in the fibrous region of the nucleolus. Many copies of the genes are present, linked together by spacer regions.
The 45S precursor is modified by methylation and undergoes a number of cleavages that ultimately produce 18S rRNA and 28S rRNA; the latter is hydrogen-bonded to a 5.8S rRNA.
18S rRNA complexes with proteins and forms the 40S ribosomal subunit.
The 28S, 5.8S, and 5S rRNAs complex with proteins and form the 60S ribosomal subunit. 5S rRNA is produced by RNA polymerase III outside of the nucleolus.
The ribosomal subunits migrate through the nuclear pores into the cytoplasm where they complex with mRNA, forming 80S ribosomes. (Because sedimentation coefficients reflect both shape and particle weight, they are not additive.)
rRNA precursors can contain introns that are removed during maturation. In some organisms, the enzymatic activity that removes rRNA introns resides in the rRNA precursor. No proteins are required. These autocatalytic RNAs are known as ribozymes.

Question 15

Explain how tRNAs are processed following transcription

Answer 15

• pre- tRNAs are cleaved and introns are removed by endonucleases. (Only some tRNAs have introns).
• tRNAs bases are modified at the same time that endonucleolytic cleavage reactions are occurring. Three modifications occur at most tRNAs:
  1) Uracil is methylated by SAM to form thymine.
  2) One of the double bonds of uracil is reduced to form dihydrouracil (causes loss of planar structure and stacking)
  3) A uracil residue (attached to ribose by an N-glycosidic bond) is rotated to form pseudouridine, which contains uracil liked to ribose by a carbon-carbon bond.