1
Q
DNA encodes genes, but itself is _____. For DNA sequences to carry out functions, it must …
A
inert; converted to RNA via transcription
2
Q
Transcription uses an enzyme called
A
RNA polymerase:
- works like DNA polymerase in many ways
- binds to DNA template and makes an RNA copy of one of two strands
- Copied strand = coding strand; other = template
- only builds RNAs 5’-3’
**Coding strand matches RNA sequence .. except T is replaced with U
3
Q
T or F. Transcription only goes in one direction
A
T, transcription from 2 DNA strand goes goes in opposite direction
4
Q
RNA polymerase core enzyme made up of 4 subunits:
A
alpha (2 copies)
beta
beta’
and omega (ω)
5
Q
Holoenzyme
A
has the 4 subunits plus sigma factor
6
Q
Transcription will generally continue until RNAP encounters…
A
a transcriptional terminator; RNAP then dissociates from DNA, stops making RNA and releases transcript
7
Q
Intrinsic (rho-independent) terminators
A
form when RNA hairpin structures form, followed by a string of “U” residues. U residues act as a pause signal for RNAP – formation of hairpin forces RNAP off template
8
Q
Rho-dependent terminators
A
A protein called Rho binds RNA as it is being transcribed and causes RNAP to dissociate after it encounters certain sequences
9
Q
Transcriptional initiation is guided by DNA sequences called
A
promoters
10
Q
What dictates whether a sequence acts as a promotor/activates a promoter
A
binding of sigma factors and the activity of regulatory proteins
11
Q
Three major classes of RNAs and other non-coding RNAs with a range of functions (often regulatory):
A
- Messenger RNA: converted to protein via translation
- Transfer RNA: functional RNAs, used in translation process
- Ribosomal RNA: functional RNAs, used in translation process
12
Q
Open Reading Frames (ORF)
A
sequences that are translated into proteins
13
Q
mRNAs contain both
A
ORFs and UTRs
14
Q
UTRs
A
untranslated regions - parts of the mRNA transcript that are not translated into protein
15
Q
mRNAs that encodes multiple ORFs are _____________ and are called _______
A
polycistronic; operons
16
Q
Genes in an operon ar ____________
A
cotranscribed
17
Q
Transcription in Archaea
A
- similar core aspects to eukaryotes
- archaeal RNA polymerase resembles RNAP II; also recruited to promtoers using transcription factors
- also use TATA Boxes and transcription factors
18
Q
Other aspects of transcription in Archaea are more like bacteria:
A
- process less complex than eukaryotes, no 5’ cap, no poly A tail, mRNAs fo not have introns (not spliced), no nucleus
- transcription/translation coupled like bacteria
- also use operons - multiple genes encoded y one RNA (single promoter controls expression of several genes)
- basically a less complex version of eukaryotic transcription
19
Q
Primary structure of proteins
A
chain (sequence) of amino acids
20
Q
Secondary structures of proteins
A
small segments of protein adopt simple local structures (local in 3D space, not necessarily in sequence)
21
Q
Most common secondary structure elements
A
alpha-helices and beta-pleated sheets
22
Q
How are alpha-helices and beta-pleated sheets formed?
A
by hydrogen bonding in peptide backbone (amide H and carbonyl O)
23
Q
Tertiary structure of proteins
A
full 3D structure of a protein; typically includes multiple secondary structure elements arranged in different ways and other structural features as well
24
Q
Quaternary structure of proteins
A
the result of multiple different polypeptides coming together (multimeric proteins or protein complexes)
25
The individual polypeptide chains in a multimeric protein are _______. These can be identical or different
subunits; homomeric or heteromeric
26
tRNAs
convert (translate) the mRNA sequence into a protein sequence
27
Start codon
encodes the first amino acid of a particular ORF (N terminal) ; when translation begins, typically an AUG (ATG in DNA sequence) for all three domains of life
28
Alternative start codons
GUG, UUG
| ** In E. coli, 83% AUG, 14% GUG, 3% UUG
29
In bacteria, the start codon is translated to ...
N-formylmethionine (fMet)
| (a chemically modified version of methionine) using a special tRNA
30
T or F. fMet often removed from proteins after translation
T
31
The three stop codons are
UAA, UGA, and UAG
32
T or F. Archaea uses fMet as well
F, uses unmodified methionine like eukaryotes
33
Prokaryotic ribosome
70S (2 subunits) = 30S (small) and 50S (large)
| Each subunit is comprised of rRNA and ribosomal proteins
34
E. coli 30S ribosome contains
16S rRNA and 21 proteins 50S contains 5S/23S rRNA and 31 proteins ; ribosomes are large, complex machines
35
The ribosome has 3 tRNA binding sites:
- A (aminoacyl) site: where new charged tRNAs enter an recognize the codon being translated; growing peptide from P site is transferred to the amino acid carried by the tRNA (Translocation occurs)
- P (peptidyl) site: second position -- tRNA moves to P site; this tRNA transfers growing amino acid chain to new charged tRNA that has entered the A site; once complete, tRNA lacks uncharged amino acids
E (exit) site: uncharged tRNAs exit here
36
Translocation
RNA moves 3 bases (one codon)
37
During translation, the elongation process repeats until the ribosome encounters a stop codon. Once this occurs, a protein called (_) binds
release factor: releases peptide and mRNA - 30S/50S dissociate; ribosome free to begin again
38
Polysomes
The same mRNA can be simultaneously translated by multiple different ribosomes -- multiple ribosomes on a single transcript called polysomes
39
Some proteins fold spontaneously into the correct 3D conformation just based on primary sources -- others require chaperones:
proteins that help other proteins adopt their properly folded and fully active state -- all 3 domains in life require chaperones
40
Chaperones use...
ATP hydrolysis
| -> commonly enable unfolded or unstable conformations to re-fold in a controlled fashion
41
In E. coli these are major chaperones that assist in protein folding
DnaJ/DnaK, GroEL, and GroES -- essential proteins for the cell to survive and amongst the most abundant proteins in the cell
42
NarJ
different sort of chaperone; it inserts an essential cofactor -- Moco (molybdenum) -- in the enzyme nitrate reductase
43
What system do prokaryotes use to transport proteins across (and into) the cytoplasmic membrane?
translocase systems
44
Sec secretion system & twin-arginine translocase (Tat)
ubiquitous in prokaryotes -- others are more specialized
45
Sec secretion system
- recognizes a signal sequence in the first ~20 AAs of proteins -- translocates unfolded protein before it folds
- protein either pass across CM (SecA pathway) or recognized by RNA/protein complex (signal recognition particle) and inserted into membrane (SRP pathway)
* *both pathways pass unfolded protein through a membrane channel (Sec YEG translocon)
- both pathways require ATP for E and signal sequence cleaved following translocon
- Tat pathway secretes folded proteins -- proteins that must fold in cytoplasm
46
Basic idea for regulating transcription initiation
control whether or not RNA polymerase binds a promoter and initiates transcription
(more accurately, the rate at which it occurs)
47
Transcription factors
regulates RNA polymerase binding ability to initiate transcription
48
Sensing is key:
have to know which genes to turn on and off AND WHEN!! must be able to detect cues in the environment
49
Many regulatory proteins are
DNA-binding proteins
50
DNA-binding regulatory proteins have
DNA-binding domains such as HTH domain (bind major groove of DNA helix)
51
Besides DNA-binding domain, prokaryotes also have other domains with activities like:
dimerization, interacting with other proteins (such as RNAP), regulatory domains, etc.
52
DNA-binding proteins often recognize
a protein sequence ; usually somewhat flexible (DNA structure can play a role too)
53
Often DNA sequences with direct or inverted repeats are bound by
homodimers - one monomer binds each repeat, dimerization required; ensure specificity
54
Transcription factors that promote transcription vs those that inhibit it
activators vs repressors
55
Some transcription factors are regulated allosterically:
binding of an effector (usually a small molecule such as a metabolite) activates or inactivates a protein
56
Inducers
"turn on" activator proteins (or inactivate repressors)
57
Corepressors
activate repressor proteins
58
Inducible system
off by default, but can be turned on
59
repressible system
on by default, but can be turned off
60
Machinery for breaking down lactose
encoded by lac operon
61
LacI repressor protein
will bind lac Operator to prevent transcription (when no lactose present or when glucose, if preferred is there)
62
Allolactose
isomer of lactose; if lots of allolactose then lots of lactose!
63
What happens when lactose is available?
allolactose will bind LacI and inactivates it
64
Lac operon is what kind of a system
inducible (catabolic systems are often inducible)
65
Catabolite repression
If better energy source than lactose is present, then won't want to utilize lac operon
66
lac operon requires both:
lactose and LOW glucose levels
67
ppGpp
- a nucleotide-based molecule
- produced in response to amino acid starvation
- shuts down protein synthesis and induces amino acid biosynthesis in a process called stringent response
68
stringent response
shutting down protein synthesis in order to induce amino acid biosynthesis
69
Quorum sensing
- involves signaling molecules called autoinducers
| - sensing the local density of cells through secretin/detecting specific molecules -- regulation based on that info
70
Chemical communication between microbes
Quorum sensing
| -> bacteria, archaea, and eukaryotic micorbes
71
This is used to coordinate group behaviours like biofilm formation, virulence, etc.
Quorum sensing
72
AHLs
(N-acyl-homoserine lactones) quorum sensing signaling molecules
73
Quorum sensing was first discovered in
Vibrio fischeri, a symbiont of the Hawaiian Bobtail Squid ... produces luminescent protein only when present at high concentrations in specialized light producing organ of squid
74
Two-component regulatory system
- common form of gene regulation in bacteria
- form of signal transduction
- uses two proteins: sensor kinase & response regulator
75
Sensor kinase
- usually in CM
- senses specific signal that activates kinase activity (phosphorylates response regulator)
- in absence of signal, it dephosphorylates response regulator
76
Response regulator
- when phosphorylates, becomes active
| - binds DNA to regulate expression of target genes (activator/repressor)
77
Transcriptional silencing
very tightly shutting off expression of genes by altering the genome structure at promoter regions
78
Best known transcriptional silencer
H-NS (E. coli, Salmonella, other gram _ bacteria)
79
Small, highly abundant nucleoid-binding proteins that bind, polymerize, and alter DNA structure
Transcriptional silencers
80
In any case, H-NS prevents binds and restructures DNA to a _____ structure to prevent RNAP from...
rigid; binding the DNA and/or carrying out transcription process
81
high % AT rather than G/C residue
- typically represent horizontally-acquired DNA
| - H-NS will bind these regions of the genome
82
Counter-silencing DNA-binding activators
bind specific silenced loci and reverse effects of H-NS (re-structure DNA and/or remove H-NS)-- allow specific genes to be expressed
83
This was proposed to be important fo evolution
counter-silencing
| --> keep new genes from being expressed at inappropriate times; evolve a means of expressing them when appropriate
84
Many important virulence genes are regulated by
silencing/counter-silencing
85
Global regulators
regulate large numbers of different genes in response to a given signal or environmental cue
ex: sigma factors,allosteric regulatory proteins, two-compartment systems, etc.
86
Regulon
the complete set of genes controlled by a given regulator
87
PhoPQ
acts as a global regulator of many virulence-related processes in Salmonella
88
T or F. Despite, differences in transcription mechanisms, transcriptional regulatory systems in Achaea are often synonymous with bacterial systems
T
89
Transcriptional attenuation
regulation that involves prematurely terminating mRNA synthesis
90
RNA regulatory elements -- regulation at level of RNA (after transcription but before protein is produced)
- stability of mRNAs can be controlled --- how long before they are degraded (longer lifetime means more translation and more protein produced)
- translation efficiency - whether or not RBS is free to be bound by the ribosome
91
RNases (ribonucleases)
degrade mRNAs and nucleotides; are recycled
| --> all cells contain multiple of these
92
Regulatory RNAs
non-coding RNA whose function is to regulate gene expression
93
Hfq
- RNA chaperone essential for mediating this regulation (much more than a chaperone)
- binds to both RNAs to stabilize their interaction ; sometime recruit Rnases to mediate degradation
94
Riboswitches
ligand-binding RNAs that adopt intricate 3D structures that SPECIFICALLY bind a particular small molecule
95
"relics" from RNA world
Riboswitches -- oldest mechanism for the regulation of gene expression?
96
Regulation of protein activity
- feedback inhibition
- protein-protein interactions (one protein binds another to control its activity)
- post-translational modifications (enzyme adds chemical moiety to specific AA residue; ex: phosphorylation)
97
Proteases
degrades protein and recycles building blocks (RNA has the same mechanism)
98
Clears away and recycles misfolded proteins
Proteases
MICRB 265
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