Exam II (lecture 14-16) Flashcards
Central dogma
DNA (replication)
Transcription (reversible)
RNA
Trnaslation
Protein
ncRNA
the gene segment of a nucleic acid that carries the code for a particular protein or for a functional non-coding RNA (ncRNA)
Prescence of of 2’-OH group
DNA vs RNA
DNA: no
RNA: yes
Both DNA and RNA nucleotides are joined by
phosphodiester bonds
DNA vs RNA secondary structure
DNA: double helix
RNA: many types
Stability RNA vs DNA
DNA: stable
RNA: easily degraded
RNA secondary structure
enables RNA molecules to fold into many different shapes that lend themseleves to many different biological functions.
Helical portions of RNA have the overall geometry of
an A-form duplex
Double helical characteristic of RNA
right handed helical conformation dominated by base-stacking interactions
non-watson-crick interactions contribute to
secondary RNA structure
unusual interactions contribute to the 3D RNA folding
U:A:U base triple
C:G:C base triple
what group contributes to stabilization of 3D RNA folding
2’-OH group
Base stacking
also contributes to stability of the 3D RNA structure
transcriptome
entire set of RNA transcripts produced in a cell
transcription
Enzymatic RNA synthesis directed by a DNA template
genes have different
rates of expression
RNA polymerases
Synthesize RNA
RNA synthesis direction
5’-3’ (the template DNA is copied in the 3’ to 5’ direction)
RNA polymerase use
ribonucleoside 5’-triphosphates (rNTPs) to syntehsize RNA complementary to the template
RNA polymerase adds nucleotides
to the 3-OH end ONLY
(same as DNA polymerases)
Does RNA polymerase require a primer?
NO
Does the product remain with the template (RNA polymerase)
NO
Is DNA or RNA synthesis more accurate
DNA is more accurate (1/10,000 bases)
Prokaryotes RNA polymerase
single RNA Pol
Eukaryotes RNA polymerase
atleast 3 RNA Pol’s
Where does the RNA polymerase attach
initiates transcription at the promoter “upstream” of the information contained in the gene
what signals the end of transcription?
terminator
Transcription unit
- sequence of nucleotides in DNA that encodes for a single RNA molecule
- promoter
- RNA coding sequence
- terminator
nontemplate =
coding = sense strand is NOT transcribed
template=
coding = antiesense strand is transcribed and complementary and antiparallel to the RNA product
promoter
RNA polymerase binding site on the DNA. Will determine which strand is going to be transcribed
template strand
may vary for different genes along the chromosome
General transcription steps
- RNA polymerase binds the ptomoter (forming first a closed complex)
- Promoter melting (open complex)
- Transcription initiated within complex
- Promoter clearance and elogation complex
- RNA pol dissociation from DNA and recycling
initation involves
binding of promoter and the formation of transcription bubble
chemical mechanism of RNA synthesis
the addition of an rNTP to a growing transcript is a Mg2+ dependent reaction that produces a 5’-3’ phosphodiester linkage.
Bacterial RNA polymerase core
5 subunits: 2alpha, 1beta, 1beta (prime), omega
- RNAPs look like “crab clows”
- Capable of RNA synthesis on a DNA template
- However
1. no specificity for promoter
2. no initation in vivo
No initation in vivo
Bacterial RNA polymerase core
sigma factor
directs the core enzyme to specific binding sites on the DNA
Core enzyme
2a, B’, B, w
Required for polymerization activity
Sigma factor + core enzyme =
holoenzyme
2a, 1B, 1B’, 1w and sigma factor
holoenzyme
required for correct initation of transcription: binding to promoter
Rpo
RNA polymerase
E. coli has several sigma factors that specify RNAP binding to particular promoters
Because different E. coli have sigma factors direct RNAP to different promoters, different sets of genes may be transcribed as “needed” by changing the sigma factor in the holoenzyme
sigma factor 70/RpoD
“housekeeping genes” expressed in all growing cells
consensus sequence
certain nucleotides that are particularly common at each position form a consensus sequence.
Bacterial promoters
How is a consensus sequence determined?
by alligning all known examples and finding most common base at each position
Consensus sequence of a sigma70 promoter
The sequence of most of the promoter is irrelevant; only short stretches of DNA are conserved
Structure/sequence identifies promoter - determines “strength”
Features of E coli promoters recognized by sigma70 (optimal promoters).
-10 region and -35 region
consensus sequences (interaction sites for sigma 70)
-10 region
5’-TATAAT-3’
-35 region
5’-TTGACA-3’
distance between -10 and -35 region
17bp
upstream promoter (UP) element
promoters of certain highly expressed genes
(bound by one alpha subunits of RNA polymerase)
mutations in the -10 and -35 regions of the promoter
affect the efficiency of RNAP binding and transcription initation
A change in just one base pair can decrease the rate of binding by several orders of magnitude
structural changes lead to open complex.
transition to open complex and to elongation requires conformational changes in RNAP and changes of its association with DNA
- place downstream duplex DNA in the active site cleft and then seprating the nontemplate and template strands.
an open complex has:
several channels, which provide access to the core of the enzyme
Initation is _ and produces short _
Primer independent; abortive transcripts
- the first 8-10 phosphodiester binds forms: high probability that the RNAP will release the transcript from the template without extending furhter
- Beyond 10 nts: the RNA becomes stable
- “release” of sigma
Transcription elongation is
continuous until termination
promoter clearance
RNA polymerase moves beyond the promoter region of the DNA to behin rapid elongation of the transcript.
transcription termination
specific sequences in the template strand stop transcription.
types of terminations
- intrinsic terminators
- Rho dependent termination
intrinsic terminatiors (p-independent)
relies primarily on structures that form in the RNA transcript
Rho dependent terminators
require rho (p) protein.
Intrinsic terminators:
2 distinguishable features
- highly conserved segment of A residues in the template that are transcribed into U residues.
- RNA transcript with self-complementary sequences - formation of a hairpin structure, centered 15-20 nucleotides before the projected end of the RNA strand.
Mechanism of intrinsic terminators
Hairpin disrupts several A-U base pairs (weak) in the RNA/DNA hybrid segment. May disturb important interactions between RNA and RNA polymerase, leading to dissociation of the transcript.
Rho (p)-dependent terminators
CA-rich sequence called a rut (rho utilization) site in the template strand (50-90 bases) long.
Rho (p) factor characteristics
- hexameric helicase
- binds to RNA very ealry in the transcription process
- RNA binding domain is the center hole of the hexamer
- ATPase activity, helicase activity
RNA that include a rut site, recruit the p helicase which
migrates in the 5’ to 3’ direction along the mRNA and seperates it from the polymerase.
Promters are recognized by
sigma70
RNA polymerase has different
intrinsic affinities for promters of different sequence
Different E.Coli sigma factors direct RNAP to different promoters
different sets of genes may be transcribed as “needed” nby changing the sigma in the holoenzyme
rpoH
sigma 32
heat shock
at 42 degrees C
induction phase - transiently increase of sigma32 levels
at 46 degrees celsius
approx. 30% of all proteion are HSPs
at 50 degrees C
sigma70 is inactivated, High levels of sigma32
at 57 degrees celsius
RNAP core is inactivated
Transcription factors
Activators and Repressors, control RNA polymerase function at a promoter
Cis acting elements
promoter, operator, activator binding site, UP element
Trans-acting factors
RNAP; repressor; activator
negative control
repressor present, transcription off
positive control
activator present, transcription on
repressor inhibits transcription
prevents or decreases expression
activator facilitates transcription
promotes or increases expression
Activation and repressors can function by
DNA looping.
regulators often work together for
signal intregation
signal integration
control of a gene by multiple regulators in response to more than one environmental signal
operon Lac
approx. 6000 bp
Operon Lac
signal integration: environmental condition
Environmental: availability of glucose and lactose
Lactose -metabolizing genes, are
under the control of an activator protein, needed for the efficienct transcription of the lac operon genes, even in the absemce of the Lac repressor.
Lactose -metabolizing genes, are
under the control of an activator protein, needed for the efficienct transcription of the lac operon genes, even in the absemce of the Lac repressor.
transcription initiation is the step most regulated
regulation at this point is the most energy efficienct, becuase it occurs before the investment of energy in mRNA
Repressors can hinder transcription binding by DNA
at a site that prevents RNA polymerase binding or by preventing closed-to-open transition of the polymerase- promoter complex (negative regulation)
Binding factors for transcription facors don’t need to be close to the transcirption start site.
Regulatory proteins that bind sites distant from the promoter exert their effects through DNA looping
Activators promote RNA polymerase binding through
cooperativity or promote formation of the open complex by causing a conformational change in the promoter or the polymerase (positive regulation)
promoters may be controlled by two or more transcription factors, allowing
inegration of signals from more than one environmental variable
small signal molecules (effectors)
allosterically regulate the function of activators and repressors
sets of genes that function in one pathway
are often controlled simultaneously.
