TRANSCRIPTION

Central dogma

gene expression

Structure

indicates function

Transcription

transfer of information from DNA → RNA

• RNA polymerase reads DNA template strand & uses complementary base pairing rules to synthesize RNA

Translation

cell synthesizes protein

Major steps of transcription

1. initiation

2. elongation

3. termination

Initation - E. coli

• sigma (part of RNA polymerase holoenzyme) binds to promoter sequences 

  • positions RNA polymerase core enzyme on the DNA template strand at the transcription start site

• RNA polymerase starts synthesizing RNA in 5’ → 3’

• sigma dissociates from the core enzyme after transcription starts

Core

RNA polymerase alone, can synthesize RNA

Holoenzyme

core + sigma factor, can recognize promotors

Elongation - E. coli

• reaction is catalyzed by RNA polymerase core enzyme

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

Polycistronic

• E. coli mRNA

• one mRNA contains the sequence information from several genes

• one mRNA encodes several proteins

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

Termination - E. coli

• intrinsic termination

• rho-dependent termination

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

Rho-dependent termination

• hairpin structure in RNA causes RNA polymerase to stall

• Rho (helicase) unwinds the RNA-DNA duplex

Prokaryotic transcription

• 1 type of RNA polymerase

  • lots of copies

• transcription and translation processes are near each other

• polycistronic mRNA (several proteins encoded in one mRNA)

• limited RNA processing

Eukaryotic transcription

• 3 different types (classes) of RNA polymerase (I, II, III)

• transcription and translation are temporally and spatially separate by nuclear envelope

• monocistronic mRNA (one protein encoded in one mRNA)

• mRNA is highly processed (5’ cap, splicing, poly A tail) before being exported from the nucleus

Polycistronic

codes for multiple proteins

Monocistronic

codes for one protein

Classes of RNA polymerase in eukaryotes

• RNA polymerase I - rRNA

• RNA polymerase II - mRNA & snRNA

• RNA polymerase III - tRNA

each are recruited to their genes by dedicated transcription factors

Initation - 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 - 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

m⁷G cap

added to the 5’ end of the mRNA

• 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

Splicing of introns

• many eukaryotic genes contain long insertions of non-coding sequences called introns

• introns

  • intervening sequence

  • transcribed but removed before translation 

  • even though they are removed, introns can contain important information

• exons

  • expressed sequence

  • transcribed and translated

Splicing

• spliceosome is made of subunits consisting of both RNA and protein 

  • subunits called snRNP (small nuclear ribonucleoprotein)

• RNA subunits base pair with RNA sequences called splice sites within the intron

  • 5’ splice site

  • 3’ splice site

  • branch point A

• loops out intron

• exons covalently bonded together

• introns released as a lariat and likely degraded

Termination - 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