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MICRB 265 > Topic 6 > Flashcards

Topic 6 Flashcards

1
Q

DNA encodes genes, but itself is _____. For DNA sequences to carry out functions, it must …

A

inert; converted to RNA via transcription

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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

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3
Q

T or F. Transcription only goes in one direction

A

T, transcription from 2 DNA strand goes goes in opposite direction

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4
Q

RNA polymerase core enzyme made up of 4 subunits:

A

alpha (2 copies)
beta
beta’
and omega (ω)

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5
Q

Holoenzyme

A

has the 4 subunits plus sigma factor

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6
Q

Transcription will generally continue until RNAP encounters…

A

a transcriptional terminator; RNAP then dissociates from DNA, stops making RNA and releases transcript

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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

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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

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9
Q

Transcriptional initiation is guided by DNA sequences called

A

promoters

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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

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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
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12
Q

Open Reading Frames (ORF)

A

sequences that are translated into proteins

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13
Q

mRNAs contain both

A

ORFs and UTRs

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14
Q

UTRs

A

untranslated regions - parts of the mRNA transcript that are not translated into protein

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15
Q

mRNAs that encodes multiple ORFs are _____________ and are called _______

A

polycistronic; operons

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16
Q

Genes in an operon ar ____________

A

cotranscribed

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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
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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
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19
Q

Primary structure of proteins

A

chain (sequence) of amino acids

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20
Q

Secondary structures of proteins

A

small segments of protein adopt simple local structures (local in 3D space, not necessarily in sequence)

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21
Q

Most common secondary structure elements

A

alpha-helices and beta-pleated sheets

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22
Q

How are alpha-helices and beta-pleated sheets formed?

A

by hydrogen bonding in peptide backbone (amide H and carbonyl O)

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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

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24
Q

Quaternary structure of proteins

A

the result of multiple different polypeptides coming together (multimeric proteins or protein complexes)

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25
Q

The individual polypeptide chains in a multimeric protein are _______. These can be identical or different

A

subunits; homomeric or heteromeric

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26
Q

tRNAs

A

convert (translate) the mRNA sequence into a protein sequence

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27
Q

Start codon

A

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

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28
Q

Alternative start codons

A

GUG, UUG

** In E. coli, 83% AUG, 14% GUG, 3% UUG

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29
Q

In bacteria, the start codon is translated to …

A

N-formylmethionine (fMet)

(a chemically modified version of methionine) using a special tRNA

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30
Q

T or F. fMet often removed from proteins after translation

A

T

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31
Q

The three stop codons are

A

UAA, UGA, and UAG

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32
Q

T or F. Archaea uses fMet as well

A

F, uses unmodified methionine like eukaryotes

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33
Q

Prokaryotic ribosome

A

70S (2 subunits) = 30S (small) and 50S (large)

Each subunit is comprised of rRNA and ribosomal proteins

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34
Q

E. coli 30S ribosome contains

A

16S rRNA and 21 proteins 50S contains 5S/23S rRNA and 31 proteins ; ribosomes are large, complex machines

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35
Q

The ribosome has 3 tRNA binding sites:

A
  • 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
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36
Q

Translocation

A

RNA moves 3 bases (one codon)

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37
Q

During translation, the elongation process repeats until the ribosome encounters a stop codon. Once this occurs, a protein called (_) binds

A

release factor: releases peptide and mRNA - 30S/50S dissociate; ribosome free to begin again

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38
Q

Polysomes

A

The same mRNA can be simultaneously translated by multiple different ribosomes – multiple ribosomes on a single transcript called polysomes

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39
Q

Some proteins fold spontaneously into the correct 3D conformation just based on primary sources – others require chaperones:

A

proteins that help other proteins adopt their properly folded and fully active state – all 3 domains in life require chaperones

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40
Q

Chaperones use…

A

ATP hydrolysis

-> commonly enable unfolded or unstable conformations to re-fold in a controlled fashion

41
Q

In E. coli these are major chaperones that assist in protein folding

A

DnaJ/DnaK, GroEL, and GroES – essential proteins for the cell to survive and amongst the most abundant proteins in the cell

42
Q

NarJ

A

different sort of chaperone; it inserts an essential cofactor – Moco (molybdenum) – in the enzyme nitrate reductase

43
Q

What system do prokaryotes use to transport proteins across (and into) the cytoplasmic membrane?

A

translocase systems

44
Q

Sec secretion system & twin-arginine translocase (Tat)

A

ubiquitous in prokaryotes – others are more specialized

45
Q

Sec secretion system

A
  • 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
Q

Basic idea for regulating transcription initiation

A

control whether or not RNA polymerase binds a promoter and initiates transcription
(more accurately, the rate at which it occurs)

47
Q

Transcription factors

A

regulates RNA polymerase binding ability to initiate transcription

48
Q

Sensing is key:

A

have to know which genes to turn on and off AND WHEN!! must be able to detect cues in the environment

49
Q

Many regulatory proteins are

A

DNA-binding proteins

50
Q

DNA-binding regulatory proteins have

A

DNA-binding domains such as HTH domain (bind major groove of DNA helix)

51
Q

Besides DNA-binding domain, prokaryotes also have other domains with activities like:

A

dimerization, interacting with other proteins (such as RNAP), regulatory domains, etc.

52
Q

DNA-binding proteins often recognize

A

a protein sequence ; usually somewhat flexible (DNA structure can play a role too)

53
Q

Often DNA sequences with direct or inverted repeats are bound by

A

homodimers - one monomer binds each repeat, dimerization required; ensure specificity

54
Q

Transcription factors that promote transcription vs those that inhibit it

A

activators vs repressors

55
Q

Some transcription factors are regulated allosterically:

A

binding of an effector (usually a small molecule such as a metabolite) activates or inactivates a protein

56
Q

Inducers

A

“turn on” activator proteins (or inactivate repressors)

57
Q

Corepressors

A

activate repressor proteins

58
Q

Inducible system

A

off by default, but can be turned on

59
Q

repressible system

A

on by default, but can be turned off

60
Q

Machinery for breaking down lactose

A

encoded by lac operon

61
Q

LacI repressor protein

A

will bind lac Operator to prevent transcription (when no lactose present or when glucose, if preferred is there)

62
Q

Allolactose

A

isomer of lactose; if lots of allolactose then lots of lactose!

63
Q

What happens when lactose is available?

A

allolactose will bind LacI and inactivates it

64
Q

Lac operon is what kind of a system

A

inducible (catabolic systems are often inducible)

65
Q

Catabolite repression

A

If better energy source than lactose is present, then won’t want to utilize lac operon

66
Q

lac operon requires both:

A

lactose and LOW glucose levels

67
Q

ppGpp

A
  • 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
Q

stringent response

A

shutting down protein synthesis in order to induce amino acid biosynthesis

69
Q

Quorum sensing

A
  • involves signaling molecules called autoinducers

- sensing the local density of cells through secretin/detecting specific molecules – regulation based on that info

70
Q

Chemical communication between microbes

A

Quorum sensing

-> bacteria, archaea, and eukaryotic micorbes

71
Q

This is used to coordinate group behaviours like biofilm formation, virulence, etc.

A

Quorum sensing

72
Q

AHLs

A

(N-acyl-homoserine lactones) quorum sensing signaling molecules

73
Q

Quorum sensing was first discovered in

A

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
Q

Two-component regulatory system

A
  • common form of gene regulation in bacteria
  • form of signal transduction
  • uses two proteins: sensor kinase & response regulator
75
Q

Sensor kinase

A
  • usually in CM
  • senses specific signal that activates kinase activity (phosphorylates response regulator)
  • in absence of signal, it dephosphorylates response regulator
76
Q

Response regulator

A
  • when phosphorylates, becomes active

- binds DNA to regulate expression of target genes (activator/repressor)

77
Q

Transcriptional silencing

A

very tightly shutting off expression of genes by altering the genome structure at promoter regions

78
Q

Best known transcriptional silencer

A

H-NS (E. coli, Salmonella, other gram _ bacteria)

79
Q

Small, highly abundant nucleoid-binding proteins that bind, polymerize, and alter DNA structure

A

Transcriptional silencers

80
Q

In any case, H-NS prevents binds and restructures DNA to a _____ structure to prevent RNAP from…

A

rigid; binding the DNA and/or carrying out transcription process

81
Q

high % AT rather than G/C residue

A
  • typically represent horizontally-acquired DNA

- H-NS will bind these regions of the genome

82
Q

Counter-silencing DNA-binding activators

A

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
Q

This was proposed to be important fo evolution

A

counter-silencing

–> keep new genes from being expressed at inappropriate times; evolve a means of expressing them when appropriate

84
Q

Many important virulence genes are regulated by

A

silencing/counter-silencing

85
Q

Global regulators

A

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
Q

Regulon

A

the complete set of genes controlled by a given regulator

87
Q

PhoPQ

A

acts as a global regulator of many virulence-related processes in Salmonella

88
Q

T or F. Despite, differences in transcription mechanisms, transcriptional regulatory systems in Achaea are often synonymous with bacterial systems

A

T

89
Q

Transcriptional attenuation

A

regulation that involves prematurely terminating mRNA synthesis

90
Q

RNA regulatory elements – regulation at level of RNA (after transcription but before protein is produced)

A
  • 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
Q

RNases (ribonucleases)

A

degrade mRNAs and nucleotides; are recycled

–> all cells contain multiple of these

92
Q

Regulatory RNAs

A

non-coding RNA whose function is to regulate gene expression

93
Q

Hfq

A
  • 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
Q

Riboswitches

A

ligand-binding RNAs that adopt intricate 3D structures that SPECIFICALLY bind a particular small molecule

95
Q

“relics” from RNA world

A

Riboswitches – oldest mechanism for the regulation of gene expression?

96
Q

Regulation of protein activity

A
  • 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
Q

Proteases

A

degrades protein and recycles building blocks (RNA has the same mechanism)

98
Q

Clears away and recycles misfolded proteins

A

Proteases

MICRB 265 (6 decks)

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