Topic 4 Flashcards
DNA is ….
inert
RNA is the….
heart of transcription
copied strand
coding strand
other strand
template strand
RNA builds
5’ to 3’
Which base is replaced in RNA and with what
T replaced with U
RNA polymerase core enzyme made up of …..
5 subunits: 2 alpha, 2 beta, w
RNA holoenzyme includes….
additional sigma unit
What does core enzyme do?
unwinds DNA and forms transcription bubble
What stops the RNA
transcriptional terminator
Intrinsic (rho-independent terminators)
Forms when RNA hairpin structures form, creating a U that pause RNAP then forces it off
RNAP
RNA polymerase
Rho-dependent terminators
protein called Rho that cause RNAP to dissociate after certain sequence
What initiates transcription
promoters
How do you is a promotor is activated
binding of sigma factors and regulatory proteins
housekeeping sigma factor
sigma70 or RpoD
Rpod
recognizes 2 sequences upstream of transcriptional start site, -35 and the Pibrew box
transcriptional start site
+1
Pribrew Box
~10 bp upstream of +1 site
Messenger RNA
converted to protein via translation
Transfer RNA
functional RNA used in translation
Ribosomal RNA
functional RNA used in translation
open reading frames (ORF)
sequences translated into proteins
untranslated regions (UTR)
parts of mRNA not translated
Polycistronic
encodes more than 1 polypeptide
Operons
cluster of genes with 1 promoter
Genes in an operon are…..
cotranscribed
5’ UTR
+1 to start codon of gene
ORF
start codon to stop codon
3’ UTR
stop codon to final transcribed residue, includes termination sequences
how is transcription different in eukaryotes (8)
- 3 polymerase
- more complex, 12+ subunits
- requires transcription factors to bind DNA and recruit RNAP
- do not use operons, each gene has own promoter
- mRNA spliced to remove introns
- 3’ polyadenylated and G cap at 5’
- mRNA transcribed in nucleus, translated in cytoplasm
- TATA box
Archea Transcription
less complex version of eukaryotic transcription
- resembles RNA pol II
- uses TATA box
- uses operons
Protein Structure?
- made of amino acids connected by peptide bonds (polypeptides)
- run N—-> C
- only get built at the C end
Name the 2 Rare Amino Acids
- Selenocysteine
2. Pyrrolysine
How are amino acids groups? Name the four groupings!
- grouped based on R-groups!
1. non-polar (hydrophobic)
2. polar
3. positively charged
4. negatively charged
Primary structure of proteins?
chain of amino acids
Secondary structure of proteins
alpha helices and beta sheets
- formed by H bonding of peptide backbone
Tertiary structure of proteins
3D structure of protein
- many secondary structures arranged together
Quaternary structure of proteins
many polypeptides coming together
===> called multimeric proteins
what are subunits in proteins?
the individual peptide chains in multimeric proteins
Identical units: homomeric
Different: heteromeric
What are protein domains?
- structural/functional segments in proteins
- can be small/large
Example of a protein domain?
Helix-turn-Helix (HTH) domains
- bind DNA (DNA binding regulatory proteins)
- found in many proteins of any Salmonella genome
what does tRNA do?
- convert/translate the mRNA sequence into a protein sequence
Each tRNA has a specific ________ that binds a particular three-base codon.
anticodon
What are tRNA synthetases?
- enzymes that ‘charge’ tRNAs (add amino acid to the CCA at the 3’ end)
How many base pairs in a codon?
3
keep going guys, we got this!!
T/F: The same tRNA can be used for two different codons.
True!
- some tRNAs work using the ‘wobble’ method by having a mismatch in the 3rd position
the typical Start codon?
typically: AUG
ATG in DNA sequence
alternative start codons?
GUG, UUG
these aren’t labelled on the codon table as start codons so we memorize them?? Uhh
What is the start codon translated to in bacteria?
N-formylmethionine (fMet) using a special tRNA
What are the ribosome subunits?
in prok.
large: 50S, small: 30S ===> total is the 70S ribosome
What mainly carries out the functions of the ribosome?
rRNA! It also catalyzes peptide bond formation
What are Shine - Dalgarno sequences?
the ribosome binding sites for translation initiation
What is used for energy in translation?
GTP!!!!
How does translation begin? (initiation)
- the ribosome binding site (shine dalgarno) binds to 16S rRNA in a random 30S ribosomal unit
- this helps robosome look for AUG codons (bona fide step start)
- fMet tRNA binds + GTP used for energy ===> 50S subunit recruited
Translation Initiation (dumbed down version)
- Shine Dalgarno finds random 30S small subunit for bon fide (which is just looking for AUG codon)
- fMet tRNA binds AUG and its ready to hunt down a 50S subunit
- 50S subunit found and bound –> ribosome is ready to translate!
The three sites of the ribosome
EPA
A site: new tRNA enters
P site: where the growing peptide chain is
e: exittttt! uncharged tRNAs leave
What is the order of steps in translation?
- Initiation
- Elongation
- Termination
Translation Termination
- ribosome encounters stop codon so a release factor binds and dissociates ribosome
What are polysomes?
Many ribosomes on one transcript.
Can RNA be translated while transcription is still ongoing?
Yup. This is coupled transcription/translation in prok.
T/F Euk. ribosomes are smaller than prok. ribosomes.
False! They’re bigger –> 40S small, 60S large
Differences between euk. and prok. translation
- in euk. transcription/translation is NOT coupled
- euk. mRNA only encodes one gene
- 5’ cap recognized in euk.
- euk. translation is slightly more complex/more regulation
Chaperones
hint: ‘changing rooms’
- help proteins to fold properly
- have controlled environment for proteins to fold properly
- use ATP hydrolysis!!!
E. coli chaperones to know
DnaJ/DnaK, GroEL and GroES
- most abundant chaperones in E.coli cells
Where are proteins synthesized in prokaryotes?
in the cytoplasm by ribosomes
How are proteins transported outside the cell if they’re required elsewhere?
reminder: proteins are impermeable to membrane
translocase systems! they help move proteins across and into cytoplasmic membrane
Two core secretion systems?
hint: sec tat
- Sec secretion system
2. twin arginine translocase (tat)
Sec secretion system
secretes UNFOLDED proteins and then the protein folds once its at the place it needs to be
Tat pathway
secretes FOLDED proteins
Regulating Transcription Initiation?
control whether or not RNA polymerase binds a
promoter and initiates transcription
Transcription Factors?
- DNA binding regulatory factors
- dictate if RNA polymerase is able to regulate transcription at a given site
Purposes of DNA binding domains?
- dimerization
- interacting with other proteins (RNA pol)
- regulatory domain
Activator
Transcription factors that promote transcription
Repressor
transcription factors that inhibit transcription
________ work by binding
DNA at promoter & recruiting
RNA polymerase (sigma factor)
to begin transcription
Activators
__________ bind DNA &
prevent RNAP DNA binding or
transcriptional initiation after it
binds.
Repressor
What does it mean to be regulated allosterically?
binding of an
effector activates or
inactivates protein
Inducers
turn on activator proteins or inactivate repressors
Co-repressors
activate repressor proteins
Inducible System?
is one that is off by default, but
can be turned on
Repressible System?
one that is on by default, but
can be turned off
What is ArgR?
repressor protein that controls the expression of an arginine
biosynthesis operon
Explain Arginine Biosynthesis?
Low arginine levels = ArgR isn’t bound by arginine, doesn’t bind DNA –> arginine is synthesized
High arginine levels = ArgR to bind
the Operator & prevent
transcription of this operon
Lac operon?
Machinery for breaking down lactose
LacI repressor protein
binds lac Operator prevents transcription
Catabolite repression
microbes eat best nutrients first, shut off secondary nutrients until needed –> not wasting energy
T/F: In the presence of glucose,
production of cAMP is
inhibited.
True!
What is required for lac operon to be expressed? (2 important things)
lactose AND low glucose levels
cAMP?
signalling molecule or second messenger
Stringent Response
ppGpp (produced in response to amino acid starvation) shuts down protein synthesis & induces amino acid biosynthesis
Quorum sensing?
sensing the local density of cells through secreting/detecting specific molecules
Autoinducers?
Quorum sensing involves signalling molecules (form of chemical communication)
Signal Transduction uses which two proteins:
Sensor Kinase and Response regulator
Sensor Kinase?
-resides in cytoplasmic membrane. -Senses specific signal -> activates kinase activity -> adds phosphate to response regulator
Response Regulator?
- When phosphorylated, becomes active
- Binds DNA to regulate expression of target genes (activator and/or repressor
Transcriptional Silencing?
very tightly shutting off
expression of genes by altering the genome structure at promoter regions
Example of transcriptional silencer:
H-NS (E. coli, Salmonella,
other Gram-negative bacteria)
Role of H-NS:
prevents binding & restructures
DNA to a rigid structure to prevent RNA
polymerase from binding the DNA
H-NS binds to …
regions of genome with high AT%
Counter-silencing?
DNA-binding activators bind specific silenced loci & reverse effects of H-NS (re-structure DNA and/or remove H-NS)
allow specific genes to be expressed
Global Regulators?
regulate large numbers of different genes in response to a given signal or environmental cue
Regulon?
complete set of genes controlled by a given regulator
Transcriptional attenuation
regulation that involves prematurely
terminating mRNA synthesis
Translation efficiency?
usually whether or not
RBS is free to be bound by the ribosome
Two major RNA structures that “shut off ”
gene expression
- Formation of a stem-loop structure involving RBS
- transcriptional terminator
Two major RNA structures that “shut off ”
gene expression
- Formation of a stem-loop structure involving RBS
- transcriptional terminator
Ribonuclease (RNase)
degrade mRNA
sRNA?
- base-pair to target mRNA(s) to regulate half-life and/or translational initiation
- affects RBS availability and/or RNase targeting
Hfq?
RNA chaperone that binds to both RNAs to stabilize their interaction
Riboswitches?
- ligand-binding RNAs –> adopt intricate 3D structures that specifically bind a particular small molecule
- allows riboswitches to sense their environment
Mechanism of Riboswitches?
Aptamer binds ligand, changes base-pairing in
the 5’UTR of mRNA to affect formation of:
(i) A stem-loop that
sequesters the RBS (blocks translation)
(ii) a transcriptional terminator
that prevents transcription of the genes.
Feedback inhibition?
An enzyme within a given biosynthetic pathway is sometimes inhibited by
the end product of that pathway
Protein-Protein interaction?
one protein binds another to control its activity
Post-translational modification
enzyme adds a chemical moiety to a specific amino acid residue of a
protein, which alters its activity (change activity, or turn it “on”/“off ”)
Role of Proteases?
enzyme used for clearing away and recycling misfolded proteins
