the process whereby genetic information in the DNA is used to create an RNA strand of complementary sequence
genome
the sum of all genetic information contained in the DNA of an organism
transcriptome
the sum of all RNA transcripts in a cell in a given moment
every cell in your body had the same _____ but not every cell has the same ________
genome; transcriptome
RNA
Ribonucleic Acid
Answer Yes or No
1.) RNA used Uracil as one of it's bases, DNA used Thymine?
2.) RNA has a hydroxyl group on its 2' carbon, DNA does not
3.) RNA has a hydroxyl group on its 3' carbon, DNA does not
4.) DNA has a phosphate group on its 5' carbon, RNA does not
1.) yes
2.) yes
3.) no
4.) no
RNA versus DNA structures
DNA can form double helix which is very regular and very stable
RNA is single stranded
RNA can also form complex secondary and tertiary structures, giving it functional capabilities
RNA transcripts can serve as carriers of information from ____ to _____
DNA; Protein
mRNA
messenger RNA
contains amino acid coding information proteins
only 3-5% of total RNA
tRNA
transfer RNA
helps in protein synthesis
15-20% of total RNA
rRNA
ribosomal RNA
helps to catalyze protein synthesis reaction
~80% of total RNA
other non-coding RNAs
smaller nuclear RNAs (snRNA)
long non-coding RNAs (IncRNA)
Micro RNAs (miRNA)
Summary of Transcription (4 steps described)
1.) RNA polymerase binds to double stranded DNA
2.) DNA opens up to expose single strands
3.) RNA polymerase read template strands and adds ribonucleotides (NTPs) to growing RNA chain
4.) DNA reforms double strand and exits, RNA chain exits
RNA transcription used the DNA _____ _______ as a template for transcription
template strand
Both the RNA transcript and the coding strand are the _____ ______ of the template DNA strand
reverse complement
The ____ ______ sequence is identical to the RNA sequence
coding strand
Structure of bacterial RNA polymerase core
5 subunits: β, β', ω, two copies of α
Bacteria have __ RNA polymerase while eukaryotes have ___ RNA polymerase
1;3
RNA Pol ___ is most similar to bacteria
II
Describe the transcription bubble
DNA duplex is unwound into single strands for a moving “bubble” of about 17 base pairs
Inside this bubble, the polymerase reaction occurs and the growing RNA chain is paired with the template strand to form a DNA/RNA hybrid (about 8 bp long)
As RNA polymerase moves through the DNA, it rotates the DNA, creating supercoils both in front and behind it
Supercoiling must be relieved by topoisomerases
sigma factor
bacterial protein that links RNA polymerase to the transcriptional start site at the front of a gene
bacterial equivalent of transcription factors
sigma factors control which genes get transcribed and when
common sigma factors and their functions
σ70- housekeeping genes, most genes
σ38- starvation genes
σ32-heat shock
σ54- nitrogen uptake and metabolism
RNA Holoenzyme
RNA polymerase "core" + Sigma Factor
How does Sigma factors know where to find on DNA?
promoters
Promoters
Region of DNA at the start of a gene, whose sequence determines where transcription begins and when
Region where RNA polymerase binds (around 100bp, -70 to +30)
Consensus Sequence
region in the promoter where sigma factor binds
-35 and -10 regions both contain consensus sequences
Upstream Promoter (UP) element
AT-rich sequence, recognized by a subunit of RNA polymerase
Increase binding and transcription
How do we know where sigma factors bind?
footprinting assay
footprinting assay
bind proteins (Sigma Factors) to DNA the chop up the DNA, and see which regions are protected
Footprinting Assay Steps 0-3
0.) mix osmY DNA fragments radiolabeled on one end with sigma factors (or nothing)
1.) Add Dnase I to cleave DNA
-light treatment ensures only 1 cut per fragment
-randomly cleave DNA into different sized fragments
2.) Remove bound protein and denature DNA
-leaves naked, radiolabeled single DNA strand
3.) Run on a gel and visualize by exposure to x-ray film
(regions of DNA bound by sigma factor/RNA pol will not be cut)
Bacterial Transcription
Initiation
Elongation
Termination
Transcription Initiation, Part 1
RNA polymerase holoenzyme (core + sigma factor) binds to DNA at promoter consensus sequence. This complex is initially closed.
A structural change causes the DNA to melt (separate into two strands), and enter the polymerase active site, creating an transcription-competent open complex.
Sigma factor is blocking the exit channel for the RNA transcript
This process is spontaneous, reversible, and requires no energy
Mechanism of the Polymerase Reaction
The 3’-hydroxyl group makes a nucleophilic attack on the α phosphate of the incoming rNTP, with the concomitant release of pyrophosphate
Transcription Initiation, Part 2
RNA polymerase does not require a primer.
NTPs are added quickly, but the structure is unstable and will spontaneously stop and leave the channel. This is called abortive initiation.
Abortive initiation occurs frequently. If so, the process begins again until about 10 bases are added, stabilizing the transcript.
Sigma factor is initially blocking the exit channel for RNA. The growing transcript will dislodge sigma factor from the exit channel, causing sigma factor to release from the polymerase. Once released, the complex exits the promoter and initiation is complete. This is promoter clearance.
What is the key difference between the closed and open transcription initiation complexes?
C. )
What does the nucleophilic attacking?
A. )
What is the main factor in determining whether transcription initiation completes?
C. )
Transcription Elongation:
3 Channels in the Polymerase
-DNA entry channel
-NTP entry channel
RNA exit channel
-(no DNA exit channel)
A pin region of the polymerase helps to ....
keep the two DNA strands separate whilst in the "bubble"
Two types of Proofreading
Kinetic Proofreading
Nucleolytic Proofreading
Kinetic Proofreading
Detects (via stalling) mismatched immediately after phosphodiester bond formation
Reverses the catalytic reaction (pyrophosphorolysis)
Nucleolytic Proofreading
polymerase backtracks and nuclease activity hydrolyzes bond upstream of mismatched base
RNA polymerase makes 10-100x more errors than DNA polymerase
Two ways to terminate transcription in bacteria:
1)Rho-Independent
2)Rho-Dependent
Rho-Independent
Requires two sequences in the RNA
a)Termination sequence in RNA about 15-20 nucleotides from end of RNA.
•Creates hairpin structure
b)AAA in template strand creates UUU in RNA strand
•Hairpin dislodges RNA at AAA/UUU interface
Rho-Dependent
rut site:
rho utilization site!Sequence on RNA that recruits rho
Rho-Dependent
ρ (rho) helicase:
helicase that travels along RNA transcript
Rho-Dependent
When ρ encounters rut, will hydrolyze ATP to dislodge transcript and shut down transcription
What does the pin region of the RNA polymerase do?
B. )
Which type of proofreading is essentially the reverse of the polymerase reaction?
A.Kinetic Proofreading
B.Nucleolytic Proofreading
A.)
What sequence is NOT essential for rho-independent transcription termination?
