Prokaryotic Transcription & Gene Regulation (L43)
Core Enzyme in Transcription Elongation:
Intake channel where DNA comes in Entry channel for NTP's DNA exit channel, exits and reforms into B-DNA conformation Exit channel for newly synthesized RNA/ nascent RNA 8 bp of RNA-DNA hybrid
The primary reason for gene regulation in bacteria is to respond to changes in the environment, such as nutrient availability. _______ is the most important step determining whether a gene is expressed.
Regulation of the rate transcription initiation is the most important step determining whether a gene is expressed.
Ribosomal RNAs(rRNA)
Ribosomal RNAs(rRNA) are structural components of a ribosome, the enzyme that catalyzes translation.
Messenger RNA (mRNA)
Messenger RNA (mRNA) houses a sequence of bases that encodes the primary amino acid sequence for a protein.A mRNA serves as the template for translation by a ribosome.
Rho-Independent Termination:
The termination signal resides in the nascent RNA chain. Termination occurs due to formation of a stable hairpin structure followed by a series of 7 U's when RNA Pol passes the terminator and has bping within it. 2ndary structure will pull on RNA-DNA bp, causing RNA Pol to pause, and weak A-U bp so DNA/ RNA unstable, so RNA dissociates
Positive Regulation of lac operon
cAMP receptor protein (CRP) binds DNA in the presence of its inducer, cAMP. The CRP binding site is located upstream of the lac promoter.
Sequence-Specific DNA Binding Proteins: The molecular basis of DNA sequence-specific binding by a DNA binding protein to DNA are _________ DNA binding proteins read the genetic info through the ____ a-recognition helix: This "recognition helix" participates in ________ Prokaryotes predominant DNA binding domain (DBD) motif is
chemical bonds between amino acids in a recognition helix and the base pairs. formation of chemical bonds between the protein and bp form chemical bonds with the bp, and is positioned within the major groove in hydrogen bonds and van der Waals interactions with the base pairs. the helix-turn-helix
Promoters and terminators are Primary transcript: In bacteria, primary transcript used as
cis-acting elements everything transcribed from the promoter to the terminator mRNA without future modification
Structural Genes of the lac Operon: Transcription results in ____ _____ is the preferred carbon source for E. coli, so it will be consumed ahead of lactose. In the absence of glucose, _______ b-galactosidase catalyzes two reactions:
contains multiple genes involved in the metabolism of lactose polycistronic mRNA that will be used to make 3 lactose proteins Glucose adenylate cyclase produces cAMP. --lactose → galactose + glucose and --lactose → allolactose
Operons are ORFs encoding proteins are arranged
coordinately regulated gene clusters, contains multiple genes and multiple ORFs, but have 1 promoter and 1 terminator so results in polycistronic mRNA with multiple ORFs each encoding a diff protein 5' → 3' in a transcription unit.
Genes and operons that are regulated are equipped with an ___ Activators: Repressors: In bacteria, both activators and repressors have _______
inducible promoter that can be turned on and off. transcription factors that increase the rate of transcription from a promoter. transcription factors that decrease the rate of transcription from a promoter. helix-turn-helix DNA binding domains.
RNA is a ___________ --a sequence always read _______ --a ________ backbone. --_________in place of thymine(T). --intramolecular base pairing that yields ________
linear single-stranded polynucleotide chain with: --a sequence always read 5'→3'. --a ribose-phosphate backbone. --uracil(U) in place of thymine(T). --intramolecular base pairing that yields complex secondary and tertiary structures.
RNA polymerases use essentially the same mechanism as DNA polymerases, but ___ Substrates:
no proofreading exonuclease activity, so error prone, not big deal b/c RNA makes proteins that can be turned over, but not DNA Substrates: ribonucleoside 5' triphosphates (NTP)
An activator binds to a An activator recruits RNA polymerase to a weak promoter through
positive regulatory element located upstream of a promoter. protein-protein interactions with the a-subunit of polymerase
Transcription starts at a ________ and ends at a ________. The finished RNA molecule is a _________ The transcription start site of a gene is always _____
promoter; terminator primary transcript +1, no zero positions
In bacteria the ___________ is the major determinant of gene expression. ___________ directly affects the rate of transcription initiation from a constitutive promoter. "Strong promoters" have ________ "Weak promoters" have_______ A mutation in a promoter that moves away from the consensus sequence ______ the rate of transcription initiation.
rate of transcription initiation Similarity to the consensus sequence very high sequence identity with the promoter consensus sequence. several base differences. decreases the rate of transcription initiation.
Homodimeric DNA binding proteins are common, there is a _____ As a result, this protein recognizes a ________. Why dimers and oligomers?
recognition helix on both subunits. palindromic sequence improved specificity--improved stability
Rho-Dependent Termination:
rho is a hexameric protein that binds a nascent RNA chain at a rut (Rho Utilization termination) site.--rho is a RNA helicase that translocates along the RNA 5'→3' using ATP hydrolysis. But RNA Pol too fast, but slows down once it starts making 2ndary structures. Rho catches up and reaches RNA Pol, unwinds the DNA-RNA hybrid, releasing the RNA
DNA Double Helix: Regulated gene expression is dependent on ___ Inducible promoters=______ Transcription factor: Sequence independent binding proteins interact with _____
sequence-specific binding of proteins called transcription factors to DNA.20 transcription factors: can be turned on and off by transcription factors binding regulates gene expression in sequence-specific manner sugar-Pi backbone
The helix-turn-helix motif is defined by Relationship: The binding of a protein to DNA often results in an ______
two alpha helices, the 2nd is the recognition helix AAs stick out from a-helix and form bonds with bp within major groove "induced bend" in the axis of the DNA.
Transcription Elongation: Rate can be slowed by
1) Dissociation of sigma factor yields core enzyme allowing RNA polymerase to complete"promoter clearance."--NusA protein replaces sigma. 2) Elongation rate accelerates to 50-90 nt/sec formation of RNA 2ndary structures 3) Core enz. transcribes until reaches terminator, so releases RNA Pol, and is recycled to be used again
Transcription Initiation:
1) sigma 70 binds to RNA Polymerase to form holoenzyme 2) Holoenzyme binds at promoter: closed complex 3) Closed complex forms open complex with strand separations, which forms transcription bubble 4) At this point, begin synthesis, de novo without promoter. begin with 2 NTPs, so at 5' end have 3 Pi's 5) Holoenzyme cont. transcription for 1st ten nucleotides at 1 nt/ sec.
1) A gene is a _______. It has some function in theorganism. Most genes encode the information needed to make a protein. A gene includes ______________ 2) ORF: 3) ORF begins at a _______ to _______ with no breaks, results in a _______, which in bacteria is the same as a _______. 4) ORF/ Cistron: sequence in mRNA that _____
1) unit of heredity; the DNA encoding the protein and the regulatory elements needed for its transcription. 2) Open reading Frame: coding sequence that contains all the instruction for making a protein 3) promoter/transcription start site to terminator; primary transcript; mRNA 4) encodes primary sequence of a protein, which will fold up to form protein
E. coli has only ______ RNA polymerase (465 kD), and it catalyzes transcription only in the ______, but can exist in 2 forms: ______ The catalytic mechanism is similar to a _______ RNA polymerase holoenzyme is responsible for _______ RNA polymerase core enzyme (a2bb'w) carries out _____
1; 5'→3' direction; holoenzyme and core enzyme DNA polymerase. transcription initiation and synthesis of first 10 nucleotides of an RNA chain. transcription elongation- does not have sigma subunit, diff. between holoenzyme and core enzyme
For example, the E. coli atp operon has _____ encoding the subunits of F1F0ATP synthase. F1F0ATP synthase is needed all of the time, so the genes of the atp operon are examples of
9 ORF's housekeeping genes b/c constantly transcribed b/c cell needs proteins all the time, typically not regulated, and never repressed. have constitutive promoter, available for transcription all the time.
Promoter: RNA Polymerase is a Bacterial promoter sequences are located on the ______ Sequence at the _______ Region with a _______ between them The bacterial consensus promoter: TTGACA-N -TATAAT-N CAT Most transcripts (RNAs) start with a _________
A promoter is a cis-acting element in the genome where RNA polymerase binds to initiate transcription. trans-acting element coding strand the -35 Region and the -10 Region with a spacer between the -35 and -10 Regions. TTGACA-N16-18 -TATAAT-N5-9 CAT a purine (+1) often within the sequence CAT.
Positive Regulation: Negative Regulation: Ligand can also prevents activator/repressor from binding to DNA.
Activator requires ligand "inducer" to bind DNA, conformational change increases transcription, Activators recruit RNA polymerase to a promoter. Repressor requires ligand to bind DNA, conformational changes in repressor, decrease transcription. Repressors inhibit transcription by RNA polymerase. Only when concentrations of ligands in cytoplasm lowers, will the ligand dissociate from activator/repressor, that will then allow it to bind
CRP binds as a ______ CRP is an _____ and its inducer ____ is present only under conditions of __ CRP is a _______, _____ protein. CRP-cAMP binds _____- lac promoter is _______ CRP recruits
CRP binds as a Dimer activator; cAMP; low glucose. homodimer; helix-turn-helix protein. CRP-cAMP binds 5' to the promoter. is "weak" because it differs from the consensus promoter. CRP recruits RNA polymerase to the lac promoter.
E. coli has _______ Sigma 70:
E. coli has Seven Sigmas Subunits recognizes promoters with the consensus sequences, recognizes constitutive promoters for housekeeping genes and inducible promoters (turn on and off)
Exposed Chemical Groups in Base Pairs
Each base pair presents a unique set of chemical groups in the major groove. CG and AT pairs can be distinguished in the minor groove. In major can tell all 4 apart
RNA Transcript Resembles the ______ The DNA template strand is said to be the __________ of the DNA coding strand and the RNA primary transcript. RNA has same sequence as The coding and template strands are defined for _______
RNA Transcript Resembles the Coding Strand "reverse complement" coding strand, only diff. is U instead of T each individual gene.
Holoenzyme consists of the a2bb'ws subunits: Subunit sigma subunit _______ Subunits a2(40 kD) are essential for _______ Subunits b(155 kD) and b' (160 kD) _____ Subunit w _______
Subunit sigma subunit recognizes a promoter. for enzyme assembly and are involved in interaction with activators. form the catalytic core. Subunit w provides structural stability.
lac Operon has _____ Operator Sites and lac repressor has _____- O1 at lac promoter is the _______ Both O2 and O3 are low affinity sites because ______ the lac repressor binds to ______ Occupancy of O1 site is responsible for A mutation destroying O1results in a
Three Operator Sites; 2 binding sites, so can bind 2 operator sites highest affinity binding site. of differences from consensus lac repressor recognition sequence. the lac repressor binds to O1 and either O2or O3 repressor activity loss of regulation of the lac promoter.
Transcription:
Transcription is the process of DNA template-dependent RNA synthesis and is catalyzed by RNA polymerase.Transcription begins at a promoter and ends at a terminator.
Transfer RNA (tRNA)
Transfer RNA (tRNA) carries an amino acid into the 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.
Bacterial promoters are inherently in the _______, so most prokaryotic gene regulation is ________. Negative regulation primarily results from_________ A repressor binds to an operator either ________ A repressor bound atop the promoter sequence _______ Repressor downstream
active state available for transcription, so most prokaryotic gene regulation is negative regulation. from a repressor binding to a site proximal to an inducible promoter. within the promoter or downstream of the promoter. blocks RNA polymerase from binding to the promoter. inhibits promoter clearance
Allolactose: The lac operon has three genes: The lacI gene is located _______ and encodes _______ lac repressor binds the operator in ______ and blocks ______ The operator site is located ______ When allolactose present,
alters ligand that effects regulation of operon lacZ, lacY, lacA. upstream of the lac operon; the lac repressor. in the absence of allolactose (or IPTG); RNA Pol from moving past promoter at the transcription start site. it binds to lac repressor and inhibits it from binding
Trans-acting factors: Cis-acting elements are
are diffusible, so they can function at multiple sites in a genome. Usually, trans-acting factors are DNA binding proteins (i.e. transcription factors) Cis-acting elements are closely tied to the gene. Typically, a cis-acting element is a DNA sequence that trans-acting factors bind to
Template strand: Coding strand: Moving in 3'->5' direction, away from transcription start site: Moving in 5'->3' direction, away from transcription start site:
tells RNA polymerase which nucleotide to add to newly synthesized RNA identical to the RNA strand numbers get smaller: upstream downstream; numbers get bigger
lac Repressor is a _____ All four subunits have ______ The inducer binding pocket is located ____
tetramer Four identical subunits: a "homotetramer" forms a "dimer of dimers". helix-turn-helix motifs for sequence-specific binding to two separate palindromic operator sites. between globular domains far from the DNA binding domain.
