Genetics Exam 1 (DNA Transcription)

Lectures

What are the major steps of transcription (3)

1. Initiation

2. Elongation

3. Termination

Initiation in Prokaryotes (ecoli)

Sigma, subunit of RNA polymerase holoenzyme) positions RNA polymerase core enzyme on the DNA template strand at the transcription start site and disassociates after transcription starts

Elongation in prokaryotes (ecoli)

• Reaction is catalyzed by RNA polymerase core enzyme

• NTPs added to the 3’ end of the nascent (new) RNA

Termination in prokaryotes (ecoli) (2 mechanisms)

1. Intrinsic termination

• Hairpin structure in RNA causes RNA polymerase to stall

• RNA-DNA duplex dissociates due to a weak dissociation between the RNA and DNA

2. Rho-dependent termination

• Hairpin structure in RNA causes RNA polymerase to stall

• Rho (helicase) unwinds the RNA-DNA duplex

What does polycistronic means and what does it allow for

• one mRNA can encode several proteins

• Allows for tight regulation of protein synthesis for proteins that are in the same pathway

Initiation in Eukaryotes

• General transcription factors bind to the core promoter

  • GTFs are named according to the polymerase with which they interact

  • Ex: TFIIA

• GTFs recruit RNA polymerase to form the preinitiation complex (PIC)

  • RNAP cannot locate a gene on its own

  • Recruitment of polymerase by GTFs is increased by activators bound to enhancer sequences (we’ll revisit this when we discuss gene regulation)

Elongation in Eukaryotes

• RNAP catalyzes NTP addition to the 3’ end of the nascent RNA strand (much like in prokaryotes)

• During transcription

  • 5’ m⁷G cap is added to the mRNA

  • Introns are spliced out (exons are spliced together) to form one continuous coding sequence

What is the purpose of m7G cap

• Attached to mRNA by a 5’-5’ linkage when mRNA is 20-30 nucleotides long

• Necessary for translation

• Protects RNA from 5’ to 3’ exonucleases

What are the subunits of spliceosome called

snRNP which base pairs at:

• 5’ splice site

• 3’ splice site

• Branch point A

which:

• Loops out intron

• Exons covalently bonded together

• Introns released as a lariat and likely degraded

Termination in Eukaryotes

• mRNA is cleaved at the polyA cleavage site

• mRNA is released from the transcription bubble

• mRNA is polyadenylated

  • About 250 adenine nucleotides (ATPs) are added to the 3’ end of the RNA by PolyA Polymerase 

    • Called the polyA tail 

  • PolyA tail is coated with protein

  • Necessary for translation

  • Protects 3’ end of mRNA from 3’ exonucleases that would otherwise degrade it

Draw transcription bubble

mRNA has the sequence of 5’-AUGUACGGAGGGUAG-3’, What is the sequence of the DNA template strand?

coding: 5’- ATG TAC …. TAG - 3’

noncoding: 3’ TAC ATG…. ATC-5’

answer:

5’- CTA CCC …. CAT-’3

Sigma subunit of bacterial RNA polymerase

• bind to bacterial gene’s promoter

• is required for RNA polymerization for holoenzyme but not for core

• Is NOT required for termination of transcription

• Is NOT required for ribosomal binding

• Is part of holoenzyme and not the core enzyme

What are the four major differences between prok. and euk. transcription

complete the table

Top row: rRNA, nucleoslus, mitchondria, and chloroplast

middle row: RNAP II, mRNA+snRNA

bottom row: tRNA, nucleoplasm

3 main processing steps for euk pre-mRNA to become mature mRNA

1.) Capping of 5’ end, conected with 5’ to 5’ bond

2.) Splicing out intron, splicing tgt extrons

3.) Polyadenylation of 3’ end, adding PolyA tail

Affects snRNP binding, intron is not spliced out as it will less like complementary base pair

all