Exam 2 Flashcards
transcription
the synthesis of RNA complementary to a DNA template
translation
the ribosomal synthesis of proteins based on triplet codons present in mRNA
messenger RNA, or mRNA
an RNA molecule that encodes a protein
RNA polymerase
an enzyme that produces an RNA complementary to a template DNA strand
RNA products of transcription in bacteria can be
monocistronic or polycistronic
Core polymerases
required for the elongation phase
αI, αII, β, β’, ω
In bacteria, the RNA polymerase is made up of
Core polymerase and sigma factor
holoenzyme
the core polymerase and sigma factor when together
sigma factor
helps the core enzyme detect the promoter, which signals the beginning of the gene. A single bacterial species can make several
“housekeeping” sigma factor in Escherichia coli
sigma-70
promoter
a noncoding DNA regulatory region immediately upstream of a structural gene that is needed for transcription initiation
sigma-70
what does it do
recognizes consensus sequences at the -10 and -35 positions, relative to the start of the RNA transcript (+1). Keeps essential genes and pathways operating
phases of transcription
initiation, elongation, termination
initiation of transcription
RNA pol holoenzyme binds to the promoter followed by melting of the helix and synthesis of the first nucleotide of the RNA
elongation of transcription
sequential addition of ribonucleotides from nucleoside triphosphates (the RNA chain is extended)
termination of transcription
RNA pol detaches from the DNA after the transcript is made
RNA polymerase holoenzyme forms a loosely bound ____ with DNA
initiation of transcription
closed complex
the closed complex must become an open complex through
initiation of transcription
the unwinding of one helical turn
RNA polymerase in the open complex becomes ____ to DNA to begin transcription
initiation of transcription
tightly bound
the first ribonucleoside triphosphate of the new RNA chain is
initiation of transcription
usually a purine (A or G)
the original RNA polymerase continues to move along the template, synthesizing RNA at
elongation of transcription
~45 bases/sec
the unwinding of DNA ahead of the moving complex forms a
elongation of transcription
17-bp transcription bubble
positive supercoils ahead are removed by
elongation of transcription
DNA topoisomerases
all bacterial genes use one of two known transcription termination signals:
Rho-dependent and Rho-independent
Rho-dependent
relies on a protein called Rho and a strong pause site at the 3’ end of the gene
Rho-independent
requires a GC-rich region of RNA, as well as 4-8 consecutive U residues
Rifamycin B
effect on transcription
selectively binds to the bacterial RNA pol and inhibits transcription initiation
antibiotics
fundamental criteria
must kill or retard the growth of a pathogen, and they must not harm the host - selectivity
Actinomycin D
effect on transcription
nonselectively binds to DNA and inhibits transcription elongation
RNA differences to DNA
- usually single-stranded
- contains ribose sugar
- uracil replaces thymine
- often folds back on itself to form complex hairpins and other secondary structures
RNA stability is measured
in terms of half-life
average half-life of mRNA
1-3 minutes
rRNA (ribosomal RNA)
an RNA molecule that includes the scaffolding and catalytic components of ribosomes
tRNA (transfer RNA)
an RNA that carries an amino acid to the ribosome. The anticodon on the tRNA base-pairs with the codon on the mRNA. Shaped like a clover leaf (in 2D) and a boomerang (in 3D)
sRNA (small RNA)
a non-protein-coding regulatory RNA molecule that modulates translation or mRNA stability; can base-pair with target mRNA or other RNA sequences; 100-200 nucleotides; encoded by intergenic sequences
tmRNA (transfer messenger RNA)
a molecule resembling both tRNA and mRNA that rescues ribosomes stalled on damaged mRNAs lacking a stop codon
catalytic RNA
also called ribozyme. An RNA molecule that is capable of catalyzing reactions
central dogma
theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein. RNA viruses can use reverse transcriptase to make DNA from RNA
codons
nucleotide triplets. An mRNA molecule can be thought of a sentence and codons represent individual words, or amino acids
there are __ possible codons
- there are 64 possible codons
- 61 specify amino acids (includes the start codons)
- 3 are stop codons
functional regions of tRNA:
anticodon and 3’ (acceptor) end
anticodon
hydrogen bonds with the mRNA codon
specifying an amino acid
3′ (acceptor) end
binds the amino acid
aminoacyl-tRNA synthetase
an enzyme that condenses a specific amino acid with the 3’ OH group of the correct tRNA, thereby charging the tRNA
each cell has ____ aminoacyl t-RNA synthetases
20, one for each amino acid
each aminoacyl t-RNA synthetase must
recognize its own tRNA but not bind to any other tRNA. Each tRNA has its own set of interaction sites that match only the proper synthetase
ribosome
a large enzyme, composed of RNA and protein subunits, that translates mRNA into protein
ribosomal subunits
30S (small subunit) and 50S (large subunit) and combine to form the 70S ribosome
70S ribosome binding sites
A (acceptor) site, P (peptidyl-tRNA) site, and E (exit) site
A (acceptor) site
the region of a ribosome that binds an incoming charged tRNA
P (peptidyl-tRNA) site
the region of a ribosome that contains the growing protein attached to a tRNA
E (exit) site
the region of a ribosome that holds the uncharged, exiting tRNA
peptidyltransferase
a ribozyme that catalyzes the formation of peptide bonds. Part of the 23S rRNA of the large ribosomal subunit
____ rRNA serves as a molecular clock
16S. Genes for ribosomal RNA are highly conserved in many different species due to their slow rate of undergoing mutations
Shine-Dalgarno sequence
(ribosome-binding site). In bacteria, a stretch of nucleotides upstream of the start codon in an mRNA that hybridizes to the 16S rRNA of the ribosome, correctly positioning the mRNA for translation.
the Shine-Dalgarno sequence is located _ of the start codon in Escherichia coli
4-8 bases upstream
start codons
AUG (90%), GUG (8.9%), UUG (1%). Code for N-formyl-methionyl-tRNA (fMet-tRNA)
Streptomycin
effect on translation
inhibits 70S ribosome formation
Tetracycline
effect on translation
inhibits aminoacyl-tRNA binding to the A site
Chloramphenicol
effect on translation
inhibits peptidyltransferase
Erythromycin
effect on translation
causes abortive translocation
translocation
the energy-dependent movement of a ribosome to the next triplet codon along an mRNA
coupled transcription and translation
ribosomes will bind to the 5’ end of the mRNA and begin translating protein before RNA polymerase has even finished making an mRNA molecule
coupled transcription and translation occurs near __
the nucleoid
translation of fully transcribed mRNA occurs at __
the cell poles
polysome
once a ribosome begins translating mRNA and moves beyond the ribosome-binding site, another ribosome can immeditaly jump onto that site. Closely packed and arranged helically along mRNA
step after translation for many proteins
a protein must be modified after translation to achieve an approriate 3D structure or to regulate its activity
a healthy cell “cleans house”
by degrading damaged or unneeded proteins by specific devices such as proteases or proteasomes
N-formyl group
protein structure modification after translation
may be removed by methionine deformylase to leave just methionine on the polypeptide. This can escape immunorecognition because fMet peptides are produced only be bacteria and mitochondria
addition of phosphoryl or methyl groups
protein structure modification after translation
can change the activity of signal transduction
adenylation
protein structure modification after translation
covalent attachment of adenosine 5’ monophosphate, can regulate the activity of enzymes such as glutamine synthetase
acetylation
protein structure modification after translation
can serve multiple functions, including protein stabilization and regulation of protein activity
lipidation
protein structure modification after translation
covalent attachment of lipids to proteins and provides hydrophobic tail that anchors lipoproteins to the cytoplasmic membrane or to the outer membrane in Gram-negative organisms
glycosylation
protein structure modification after translation
covalent addition of monosaccharides or polysaccharides to generate glycoprotein
mass spectrometry
an analytical technique that measures the mass of molecules and to assess posttranslational modifications. Molecules are ionized and sorted according to their mass-to-charge (m/z) ratio
chaperones
a protein that helps other proteins fold into their correct tertiary structure
heat-shock proteins (HSPs)
name of original chaperones. A chaperone protein whose synthesis is induced by a high-temperature stress. More resistant to heat denaturation than the average protein is
trigger factor (TF)
acts as a ribosome-associated chaperone and is the first chaperone to interact with nascent polypeptide
GroEL structure
forms a stacked ring structure with a hollow center
GroES structure
cap-like structure that fits over one end of GroEL
GroEL and GroES complex
facilitate protein folding; protein enters central cavity of GroEL, and the GroES cap helps create a controlled environment for folding
DnaK and DnaJ complex
do not form rings, act as clamp-like chaperones
DnaK
also known as Hsp70, can bind to exposed hydrophobic regions on unfolding or misfolded proteins, preventing their aggregation and assisting in proper folding
DnaJ
often works with DnaK, helps recruit substrates and regulate DnaK’s ATPase activity
degrons
degradation signal in many normal proteins that dictate the stability of a protein
N-terminal rule
type of degron; tendency of the N-terminal amino acid (leucine, phenylalanine, tryptophan, or tyrosine) of a protein to influence protein stability; cleaved by ClpAP protease
protease
enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products.
N-terminal signal sequences
a specific amino acid sequence on the amino terminus of proteins that directs them to the endoplasmic reticulum (of a eukaryote) or the cell membrane (of a prokaryote)
signal recognition particle (SRP)
a receptor that recognizes the signal sequence of peptides undergoing translation. The complex attaches to the cell membrane of prokaryates (or rough ER of eukaryotes), where it docks the protein-ribosome complex to the membrane for protein membrane insertion or secretion
FtsY
shuttles the complex delivered by SRP to the transmembrane SecYEG secretion machinery (translocon)
SecA-dependent general secretion pathway
common mechanism in bacteria for translocating certain proteins from the cytoplasm to the periplasm. Essential for proper localization of periplasmic proteins
SecB
piloting protein that captures the completed pre-secretion protein and prevents aggregationg and misfolding
SecA
takes pre-secretion protein from SecB; is an ATPase associated with the SecYEG translocon
SecYEG translocon
protein complex embedded in the bacterial inner membrane that serves as a channel for protein translocation across the membrane
twin arginine translocase (TAT)
transport complex that assembles on demand to ship fully folded proteins across the cell membrane to the periplasm; powered by the proton motive force
type I protein secretion
moves certain products directly from the cytoplasm to the environment; typically found in Gram-negative bacteria
ABC transporter
short for ATP-binding cassette transporter; membrane protein complex that plays a crucial role in the active transport of various molecules across biological membranes; simplest of protein secretion systems; make up type I protein secretion
type I systems all have three protein components
- an outer membrane channel
- an ABC transporter protein at the inner membrane
- a periplasmic protein lashed to the inner membrane
cells do not express every gene at maximal level under all conditions for a number of reason
- physical space limitations
- energy and resource conservation
- contradictory functions
gene regulation occurs at multiple, often interwoven levels
- alteration of DNA sequence
- control of transcription
- control of mRNA stability
- translational control
- posttraslational control
control at the DNA level is ___
the most drastic and least reversible
control at the protein level is ___
the most rapid and most reversible
transcriptional regulators (activators and repressors)
regulatory proteins that control gene transcription by binding to specific DNA sequences; activity influenced by ligand molecules whic can bind and modulate their function
two-component signal transduction system (TCS)
a message relay system composed of a sensor kinase protein and a response regulator protein that regulates gene expression in response to a signal (usually extracellular)
sensor kinase
a transmembrane protein that phosphorylates itself from ATP in response to an extracellular signal and transfers the phosphoryl group to a receiver protein
alternative sigma factors (RpoS, or σS)
can recognize and bind to specific promoter sequences, directing RNA polymerase to transcribe specific sets of genes in response to certain conditions; in many Gram negative cells
riboswitch
a secondary structure with some mRNA transcripts that can interact with metabolites or antisense RNA molecules, change structure, and affect production or translation of mRNA by folding into 3D structures; found upstream of coding sequence;
second messenger
a regulatory molecule produced in response to primary signal; typically affect expression of numerous genes
___ is a major site of regulatory control in bacterial cells
transcription initiation
___ control initiation of transcription at gene promoters
DNA-binding regulatory proteins
regulatory proteins
a protein that can bind DNA and modulate transcription in response to a metabolite; controls transcription initiation
the sequence of DNA affects the ___
binding affinity of regulatory proteins
repressor
a regulatory protein that can bind to a specific DNA sequence and inhibit transcription of genes; prevent gene expression (repression) by binding to DNA sequences called operators
activators
a regulatory protein that can bind to a specific DNA sequence and increase transcription of genes; stimulate gene expression by contacting an RNA polymerase positioned at a nearby promoter
operators
a region of DNA to which the repressor protein binds; usually located near promoters
inducer
ligand that stimulates transcription of genes by changing the DNA-binding properties of a regulatory protein; binds to a repressor protein making it lose the ability to bind the operator and blocks transcription; binds to an activator protein making it gain the ability to bind the DNA and the RNA polymerase to stimulate transcription
response regulator
a cytoplasmic protein that is phosphorylated by a sensor kinase and modulates gene transcription depending on its phosphorylation state; controlled by covalent modifications
Jacques Monod and François Jacob
French scientists who proposed the revolutionary idea that genes could be regulated in 1961; noticed that enzymes used to metabolize lactose in E. coli were inducible, whereas those for glucose were constitutive
lactose
disaccharide sugar made of glucose and galactose that can be used as a carbon and energy source
lactose metabolism requires two components:
- lactose permease (LacY): imports (transports) lactose into the cell
- β-galactosidase (LacZ): cleaves lactose into the monosaccharides glucose and galactose
lac operon promoter
PlacZYA; serves as binding site for RNA polymerase to initiate transcription
lac operon operator
lacO; DNA sequence upstream of promoter and serves as binding site for lac repressor
lactose operon repressor
LacI; regulatory protein situated immediately upstream of the operon
in the ___ LacI binds as a tetramer to the operator region and ___ transcription by preventing open complex formation by RNA polymerase
absence of lactose, represses
in the ___ of lactose, β-galactosidase (LacZ)
cleaves and rearranges lactose to make ___
presence and inducer allolactose
allolactose
inducer; binds to LacI and reduces its affinity for the operator
cyclic AMP (cAMP)
governs the level of lacZYA transcription in E. coli by binding to a dimeric regulator called CRP; accumulates when a cell is starved for carbon
cAMP-CRP complex
binds to a DNA sequence located 60bp upstream; causes DNA to bend, allowing CRP to interact directly with alpha subunit of RNA polymerase bound at the PlacZYA promoter to activate transcription
maximum expression of the lactose operon requires ___ and ___
removal of the lactose repressor (LacI) and the binding of an activated cAMP-CRP complex
catabolite repression
an operon enabling the catabolism of one nutrient is repressed by the presence of a more favorable nutrient; ex: glucose is favored over lactose
diauxic growth
a biphasic cell growth curve caused by depletion of the favored carbon source and a metabolic switch to the second carbon source
inducer exclusion
the ability of glucose to cause metabolic changes that prevent the cellular uptake of less favorable carbon sources that could cause unnecessary induction; glucose transport by phosphotransferase system inhibits LacY and prevents lactose from entering
aporepressors
genes encoding biosynthetic enzymes typically regulated by inactive repressors; becomes active when corepressor binds to it
corepressor
binds to aporepressors and the complex binds to an operator sequence upstream of a target gene or operon to turn transcription off
trp operon
codes for enzymes involved in biosynthesis of the amino acid tryptophan
aporepressor protein (TrpR)
when tryptophan levels exceed cellular needs, excess binds to this inactive TrpR and converts it into an active repressor
regulon
collection of coregulated operons that are scattered around the bacterial chromosome
protease ClpXP
orchestrates the stationary phase-dependent accumulation of σS by modulating its degradation
when cells enter stationary phase degradation of σS ___
stops
anti-sigma factor
proteins that inhibit activity not by degradation, but instead by binding directly to specific sigma factors and blocking their access to the RNA polymerase; release is important for spore formation and flagella construction
anti-anti-sigma factor
can neutralize anti-sigma factors
attenuation
a regulatory mechanism in which translation of a leader peptide affects transcription of an operon’s downstream structural genes
asRNA (cis-antisense RNA)
molecules are transcribed from
the nontemplate strands of mRNA-encoding genes; affects RNA stability; 700-3000 nucleotides
Guanosine tetraphosphate (ppGpp)
a second messenger that allows cells to handle abrupt changes in nutrient availability by switching from rapid growth to slower growth.
stringent response
nutrient scarcity leaves many ribosomes idle
Cyclic di-GMP (c-di-GMP)
a second messenger used by E. coli cells to
transition between a motile, single-cell (planktonic) state and an adhesive multicellular (biofilm) state
autoinducer
a membrane-permeable second messenger that allows cells to regulate gene expression based on the density of cells in the environment
quorum sensing
mechanism that bacteria use to coordinate gene expression in response to changes in population density; first discovered in Aliivibrio fischeri
LuxI (in V. fischeri)
synthesizes autoinducer, which diffuses into extracellular medium
LuxR regulatory proteins (in V. fischeri)
autoinduce reenters the cells and binds to these proteins when threshold levels are reached
LuxR-autoinducer complex
activates transcription of luciferase genes that confer luminescence
Pseudomonas aeruginosa quorum sensing
forms a biofilm in the lungs of cystic fibrosis
patients and secretes virulence factors like proteases only when it can overwhelm its host and avoid immune responses with high cell densities
Vibrio harveyi quorum sensing
uses three different, but converging, quorum-sensing systems; separate autoinducers for each system may be made by other microbial species
competent cells
can import free DNA fragments and integrate them into their genomes by recombination
competence stimulation peptide (CSP)
autoinducer produced by competent cells; activates a phosphorylation cascade that ultimately actives ComX, an alternative sigma factor used specifically to transcribe genes that establish competency
benefits of taking up foreign DNA
- imported DNA can be used as a food source
- can repair damaged chromosomes
- drive genome evolution
Agrobacterium tumefaciens which causes crown gall disease
- can transfer genes across biological domains
- contains a tumor inducing Ti plasmid that can be transferred via conjugation to plant cells
F factor replication origins:
- oriV: used in nonconjugating cells
- oriT: used during DNA transfer
tra genes
responsible for several aspects of conjugation, including pilus formation and DNA export
relaxase
plasmid codes this protein for DNA transfer and unwinds DNA
the entire chromosome takes about ___ to transfer
100 minutes
generalized transduction
can transfer any gene from a donor to a recipient cell
specialized transduction
can transfer only a few closely linked to the phage insertion site between cells
Vibrio cholerae
gram-negative, extends a type IV pilus, which can actively take up free DNA in the environment
Transposable elements transpose by one of two mechanisms:
- replicative transposition: transposable element jumps from one site to another
- nonreplicative transposition: transposable element is copied, one copy remains in original site
homologs
genes with shared ancestry have sequence similarity, or homology
orthologs
homologs found in different species, same function
