Biochem 508 Lec 7: Regulation of gene expression I

T or F: mRNA is the only kind of RNA

FALSE! Other RNAs we care about are rRNAs and tRNAs

Does cellular life have ss or dsRNA?

SS

What nucleotide base is different from RNA to DNA

Thymine is Uracil in RNA!

What is different about the ribose sugar structure on RNA from DNA?

There’s a 2’ OH group that DNA lacks (that’s why it’s called DEOXYribo.)

mRNA codes for…

proteins!

The template strand of DNA is called ___ because… On the other hand, the non-template strand of DNA is called ___ OR ___because…

• Template: antisense, RNA strand is the reverse complement of this strand

• Non-template: sense OR coding strand, RNA strand matches the seq on this strand (besides U)

RNA is synthesized from DNA in a process called __ by an __ Polymerase. What direction does RNA Pol synthesize in?

Transcription, RNA. 5’ to 3’, just like ALL polymerases

To synthesize an RNA template with seq AUGGCUC, the DNA template would have a seq of…

Reverse complement!

GAGCCAT

E. Coli RNA Pol has __ different kind of subunits, but __ total

5, 6

Nucleotide addition is a __ catalytic mechanism across polymerases!!

conserved

RNA Pol uses a __ __ mechanism and this happens via a ___ attack of the __OH group on the __ phosphate of the incoming dNTP

Two metal, nucleophilic, 3’, alpha

What residues on RNA Pol are coordinated to the 2 Mg+ ions? Why?

Asp, because it has a - oxygen.

Which domains of life only use 1 RNA Pol, and which uses multiple for producing different types of RNA?

• Bacteria and archaea: 1

• Eukaryotes: multiple! (3)

Which eukaryotic RNA Pol produces mRNA

RNA Pol II

RNA Pol __ and __mechanism are conserved across all domains of life

structure, synthesis

T or F: RNA Pols require a primer to begin synthesis

FALSE! They can start de novo!

Some RNA pols have a proof reading mechanism, but they’re different from DNA pols how?

No proofreading exonuclease domain in RNA Pols

Describe how DNA topology changes as RNA Pol translocates across the DNA strand

Overwinds the DNA ahead of it, underwinds the DNA behind it

__ are used to relax strain in DNA during transcription

Topoisomerases

What topoisomerase can relax negative supercoils? Positive?

• Negative: Topo I

• Positive: DNA Gyrase (by inducing - supercoils)

T or F: only 1 RNA Pol can work on a gene at a time

FALSE

Prokaryotic RNA Pol can/can not bind to the DNA template directly? What factors are needed to enhance sequence specificity?

Eukaryotic RNA Pol can/can not bind to DNA template directly?

• Prokaryotic: CAN, sigma factors

• Eukaryotic: CANNOT

What function do promoter sequences serve?

Recruit RNA Pol to a gene

What are the two consensus sequences/ components of a prokaryotic promoter seq

• -10: TATA Box

• -35

The -10 and -35 promoter seq in prokaryotes are recognized by __ __

Sigma factors

Regulatory sequences can be… of a promoter seq

upstream, downstream, or even overlapping

If there is a mutation in the -10 or -35 consensus sequences, how will this affect transcription?

Transcription levels will be much lower since the sigma factors bind to specific sequences

Sigma factors induce __ __ of the DNA at the -10 and -35 consensus sequences

local melting

The local melting induced by the sigma factors produces…

A bubble of ss DNA, in which one strand (template strand) will be placed inside the RNA Pol active site

Sigma factors provide ___ for RNAP binding to promoters

Specificity

What two sigma factors should we know?

• Sigma 70: important for housekeeping genes (70 kDa)

• Sigma 32: important for heat shock genes (32kDa)

What is abortive transcription?

• Sigma bound RNAP can only synthesize a few short nucleotides before terminating

• In order for continuous synthesis, RNAP needs to “escape” from the promoter and disassociate from the sigma factors

The basal rate of misincoorperating the wrong nucleotide for a polymerase is…

Every 1,000 to 10,000 bp

Do RNAPs have exonuclease activity?

NO

How do RNAPs fix errors they make during transcription, then?

When they misincorperate a nucleotide, they stall and sometimes reverse, and remove it using a two metal mechanism

What is the nucleophile in the two metal mechanism when HYDROLYIZNG phosphodiester bonds?

Water!

What are two ways that prokaryotes can terminate transcription?

Rho dependent and Rho independent (AKA intrinsic)

Rho is an…

ATP dependent helicase

In order for Rho dependent termination to happen, there must be a …

Rut site in the product RNA that Rho can bind to

Describe Rho dependent termination

1. Rho binds to rut site on RNA

2. Rho uses ATPase activity to chase RNAP, moving 5’ to 3’

3. Once it has caught up, Rho unwinds the RNA/DNA duplex in the RNAP active site, terminating transcription. It da pulls out the RNA from the active site.

Rho independent termination relies on the formation of an RNA __.

Hairpin

Describe how the RNA hairpin is formed in Rho independent termnation

1. Hairpin formation begins at the top, and completes on its way down

2. Its the RNA that’s forming the hairpin, followed by a U tract base paired to a DNA a tract

What is the purpose of NusA

Bacterial transcription factor that stabilizes RNA hairpin and prevent Rho dependent termination by blocking the binding of Rho

Causes the RNAP to pause adjacent to termination signals, because the binding of NusA to the RNA hairpin distorts the RNAP active site, inhibiting RNAP translocation

Full formation of the RNA hairpin requires.. The result is…

nucleotides that are paired with DNA, and the incorporation of these nucleotides breaks the H-bonds holding together the RNA:DNA hybrid, thus collapsing the transcription bubble