prokaryotic transcription and gene regulation Flashcards
transcription
process of DNA template-dependent RNA synthesis and is catalyzed by RNA polymerase
messenger RNA (mRNA)
houses a sequence of bases that encodes the primary amino acid sequence for a protein
- serves as a template for translation by a ribosome
transfer RNA (tRNA)
carries an amino acid into catalytic site of a ribosome. the tRNA base pairs to mRNA to ensure selection of the correct amino acid for incorporation into a nascent polypeptide chain
ribosomal RNAs (rRNA)
structural components of a ribosome, the enzyme that catalyzes translation
_____ is typically a linear single-stranded polynucleotide chain with:
a ribose sugar phosphate backbone
uracil in place of thymine
the sequence always read 5’->3’
RNA
________ base pairing is allowed when RNA base pairs with itself or another RNA molecule
G-U
internal base pairing within an RNA strand yields…
complex secondary and tertiary structures
RNA polymerase will use one strand of DNA as the ____________ strand when synthesizing RNA
template
the DNA template strand is said to be the ________________ of the DNA coding strand and the RNA primary transcript
reverse complement
the RNA transcript resembles…
the coding strand
trans-acting factors
diffusible so they can function at multiple sites in a genome; usually are DNA binding proteins; affects levels of transcription
cis-acting elements
closely tied to the gene; typically is a DNA sequence; fixed place in genome
transcription starts at a __________ and ends at a ____________. the finished RNA molecule is a ________________________
promoter; terminator; primary transcript
a gene includes the ….
DNA encoding the protein and the regulatory elements needed for its transcription
in bacteria, a primary transcript is used as…
an mRNA for translation without further modification
open reading frame (ORF)
sequence of bases that encodes the primary sequence of a protein; aka coding sequence
operons
coordinately regulated gene clusters
ORFs encoding proteins are arranged 5’->3’ in a transcription unit
use of one promoter and terminator yields a polycistronic mRNA with multiple ORFs each encoding a different protein
a _________ is a cis-acting element in the genome where ___________ binds to initiate transcription
promotor; RNA polymerase
the bacterial consensus promoter
TTGACA-N16-18-TATAAT-N5-9CAT
in bacteria, the rate of _____________________ is the major determinant of gene expression
transcription initiation
Similarly to the consensus sequence directly affects the rate of transcription initiation from a __________________
constitutive promoter (always available/on)
strong promoters
have very high sequence identity with the promoter consensus sequence
weak promoters
have several base differences
a mutation in a promoter that moves away from the consensus sequence…
decreases the rate of transcription initiation
E.coli has only one …
RNA polymerase
____________________ is responsible for transcription initiation and synthesis of 1st 10 nucleotides of RNA
RNA polymerase holoenzyme
holoenzyme consists of the ____________ subunits
a2BB’wsigma
subunit sigma
recognizes a promoter; binds to most of the promoters in the E.coli genome
subunits a2
essential for enzyme assembly and interact with activators
subunits B and B’
form the catalytic core
subunit w
provides structural stability
RNA polyermase core enzyme (a2BB’w)
carries out transcription elongation
RNA can start synthesizing RNA _________________. it will form a phosphodiester bond between 2 NTPs to being RNA synthesis. The 5’ end will contain 3 phosphate groups.
de novo (without a primer)
RNA polymerase requires a DNA template chain but does not have ….
proof reading exonuclease activity meaning there will be errors ( its okay bc RNA is degraded quickly)
NTP
ribonucleoside 5’triphosphates
holoenzyme binds to a promoter forming the _______________.
- ___ identifies the promoter and makes protein-DNA interactions with -35 and -10 promoter regions (initiation)
closed complex; sigma70
unwinding of DNA 12-15 bp forms a transcription bubble converting a closed complex to an _____________ (initiation)
open complex
______________ initiates RNA synthesis and synthesizes about 10 nt (initiation)
holoenzyme
dissociation of the sigma factor yields core enzyme allowing RNA polymerase to complete ______________
promoter clearance
__________________ rate by core enzyme accelerates to ~50-90 bases/sec (not being held back by sigma anymore)
- rate of ______________ can be slowed by formation of RNA secondary structure in the transcript
- __________________ relieve supercoiling in DNA
transcription elongation; topoisomerases
________________________ results in release of RNA and dissociation core enzyme from DNA
transcription termination
rho-independent termination
termination signal resides in the nascent RNA chain sequence; no protein needed
termination occurs due to formation of a stable hairpin structure followed by a series of 7 Us (rho-independent terminator)
hairpin formation results in only a few weak U=A base pairs between transcript RNA and DNA. DNA/RNA is unstable so the RNA dissociates, termination transcription
rho-dependent termination
requires Rho protein
rho is a hexameric protein that binds a nascent RNA chain at a rho utilization site (rut) within the RNA
rho is an RNA helicase that translocates along the RNA 5’->3’ using ATP hydrolysis
RNA polymerase pauses in response to formation of an RNA secondary structure
transcription terminates when rho contacts RNA polymerase
inducible promotors can be _________________
turned off and on
repressors
transcription factors that decrease the rate of transcription from a promoter
activators
transcription factors that increase the rate of transcription from a promoter
in bacteria, both activators and repressos have ________________ DNA binding domains
helix-turn-helix
regulated gene expression is dependent on sequence-specific binding of proteins called __________________ to DNA
transcription factors
transcription factors bind to ___________ and ____________
major and minor grooves
an ________ of the DNA binding protein is positioned within the _________ groove
a-helix; major
_______________ participates in hydrogen bonds and van der Waals interactions with the base pairs
recognition helix
in prokaryotes, the predominant _____________ motif for containing a recognition helix is called the _______________
DNA binding domain (DBD); helix-turn-helix
formation of a stable DNA-protein complex is dependent on …
additional non-covalent chemical bonds outside the recognition helix-base pair contacts
protein motifs
small regions of protein 3D structure or amino acid sequence shared among different proteins
each base pairs presents a unique set of chemical groups in the _________
major groove
CG and AT pairs can be distinguished in the __________
minor groove
transcription factors cannot tell the difference between _____ and _____ pairs in the minor groove
AT and TA
the major groove has _____ nucleotides, while the minor groove has ____ nucleotides
4;3
helix-turn-helix motif
two a helices; the 2nd helix is the recognition helix
the DNA binding domain of a bacterial transcription factor consists of a _______________
helix-turn-helix motif
the DNA binding domain is just one small region of the transcription factor
________ DNA binding proteins are common.
- there is a recognition helix on both subunits because both subunits contain a helix-turn-helix motif
- as a result, this protein recognizes a _______________
- the same sequence of bases is seen 5’ -> 3’ along both strands
homodimeric; palindromic sequence
why dimers and oligomers?
improved specificity and improved stability (more interactions)
____________ negatively regulate transcription through inhibiting transcription by ________________. _____________ from the cellular environment control the activity of _____________. Some ____________ only bind to DNA in the presence of a ligand. Some __________ only bind to DNA in the absence of a ligand.
repressors; RNA polymerase; ligands; repressors
negative regulation primarily results from a __________ binding to a site proximal to an __________________
repressor; inducible promoter
a repressor bound downstream inhibits…
promoter clearance by RNA polymerase
a repressor bound to the promoter sequence blocks…
RNA polymerase from binding to the promoter
four identical subunits: a homotetramer forms a _________________
- all four subunits have ____________ motifs for sequence-specific binding to two separate palindromic operator sites
- inducer binding pocket is located between globular domains far from the DNA binding domain
dimer of dimers
helix-turn-helix
____ at lac promoter is the highest affinity binding site.
O1
Both ___ and ____ are low affinity sites because of differences from consensus lac repressor recognition sequence
O2 and O3
the lac repressor bind to ___ and either ___ or _____
- occupancy of the __ site is responsible for repressor activity
- a mutation destroying __ results in a loss of regulation of the lac promoter
O1; O2 or O3
O1
_________ positively regulate transcription by recruiting _________________ to a ____________. __________ from the cellular environment control the activity of __________. Some only bind to DNA in the presence of a ligand while some only bind to DNA in the absence of a ligand
activators; RNA polymerase; promoter; ligands; activators
some prokaryotic gene regulation results from binding of an activator to a site proximal to an _____________________
inducible promoter
an __________ binds to a _______________________ located upstream of a promoter
- an activator recruits RNA polymerase to a weak promoter through protein-protein interactions with the RNA polymerase a subunit
ex. CRP activator
activator; positive regulatory element
____ is an ______ and its “inducer’ _______ is present only under conditions of low glucose
- ____ is a homodimeric helix-turn-helix protein
-__________ binds 5’ to the promoter
CRP; activator; cAMP; CRP; CRP-cAMP
lac promoter is weak because…
CRP recruits _____________ to the lac promoter through protein-protein interactions with the ______________________-
it differs from the consensus promoter
RNA polymerase
RNA polymerase a subunit
in the absence of glucose, adenylate cyclase produces
cAMP
________ is the preferred carbon source for E.coli, so it will be consumed ahead of _________
glucose; lactose
(negative regulation) the _______ gene is located upstream of the lac operon and encodes the ____________
- __________ binds the operator in the absence of ___________
- the _________ site is located at the transcription start site
lacl; lac repressor; lac repressor; allolactose; operator
(positive regulation) ______________________(CRP) binds DNA in the presence of its inducer, ________
- the _______________ (activator binding site) is located upstream of the lac promoter
cAMP receptor protein; cAMP; CRP binding site
