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Bacterial Physiology (BIOL 471) > Ch. 11 > Flashcards

Ch. 11 Flashcards

1
Q

What fundamental principles does the synthesis of macromolecules follow?

A
  • Modular: made up of repeating polymers
  • Dehydration reactions are involved in polymerization
  • Reactions are energetically unfavorable
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2
Q

What is transcription?

A

RNA synthesis

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3
Q

What are the 3 stages of RNA synthesis?

A
  1. Initiation
  2. Elongation
  3. Termination
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4
Q

What are the requirements for the initiation of RNA synthesis?

A
  • Promoter
  • RNA polymerase
  • Sigma factors
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5
Q

What is the promoter?

A

DNA region that RNA polymerase binds to

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6
Q

What is the function of the Pribnow box?

A
  • Aka the -10 sequence
  • It is what RNA polymerase recognizes to bind to the promoter
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7
Q

What is the function of RNA polymerase?

A

Transcribing RNA

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8
Q

What is the function of sigma (σ) factors?

A

Responsible for the recognition of promoters by RNA polymerase (bind to RNA polymerase to form the RNAP holoenzyme)

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9
Q

At what point during RNA synthesis does elongation occur?

A

After about 12 NTPs have been added

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10
Q

What happens during the initiation stage of RNA synthesis?

A
  1. Formation of the closed complex
    - RNAP holoenzyme bound to promoter region
    - DNA enters but remains dsDNA
    - Reversible
  2. Formation of open complex
    - DNA duplex unwinds at promoter region to form open complex
    - Irreversible
  3. Binding of initiating ribonucleotides
    - Transcription is usually initiated with ATP or GTP
    - NTPs enter active site through a 2º channel
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11
Q

What happens during the elongation stage of RNA synthesis?

A
  • RNA chain weakens interaction between sigma subunit and -35 region of the promoter –> sigma subunit dissociates from polymerase
  • Polymerase moves forward from promoter as it synthesizes RNA
  • Transcription bubble (~18 bases long) in DNA duplex: where elongating RNA forms RNA/DNA hybrid with template DNA strand (helps keep RNAP attached to DNA)
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12
Q

What are the 2 patterns of termination of RNA synthesis?

A
  1. Factor-independent
  2. Rho-dependent
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13
Q

Explain factor-independent termination.

A
  • Self-complementary sequence of bases
  • RNA hybridizes itself (hairpin loop formation)
  • RNA spontaneously dissociates from DNA
  • RNAP dissociates
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14
Q

Explain Rho-dependent termination.

A
  • Rho hexamer binds to rut in the RNA directly behind RNAP
  • ATP-dependent RNA/DNA helicase
  • Binds RNA and trails RNAP until it overtakes and pulls DNA:RNA hybrid apart
  • RNAP dissociates
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15
Q

What methods do bacteria use to regulate protein synthesis at the transcriptional level?

A
  • Sigma factors/anti-sigma factors
  • Operons
  • Transcription factors
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16
Q

Transcription is commonly regulated at the initiation step by proteins that affect _____, _____, and _____.

A
  • the binding of RNAP to the promoter
  • the “melting” of DNA to form transcription bubble
  • movement of RNAP along DNA (elongation)
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17
Q

Selection of promoters by RNA polymerase is dependent on the _____ that associates with it.

A

Sigma factor

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18
Q

What are the 2 families of sigma factors?

A
  1. σ-70
  2. σ-54
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19
Q

What do anti-sigma factors do?

A

Bind sigma factors and inhibit their activity

20
Q

Give an example of an anti-sigma factor (what it is, how it works, what happens to it)?

A

Ex. RseA
- Transmembrane protein with a cytoplasmic domain that binds to σE and keeps it inactive
- RseB (periplasmic protein) binds and stabilizes RseA
- σE is used to transcribe genes that are important for repairing the cell envelope when its proteins become damaged
- When cells are stressed –> OMPs become misfolded –> activate DegS
- DegS and Yael (cell membrane proteases) sequentially degrade RseA –> activate σE

21
Q

What is an operon?

A

A sequence of DNA containing a cluster of genes under the control of a single promoter
- Polycistronic mRNA

22
Q

What are the 3 basic components of an operon?

A
  1. Promoter
  2. Operator
  3. Genes
23
Q

What binds to each of the components of an operon?

A
  1. Promoter: RNAP binds
  2. Operator: repressor protein binds
  3. Genes: N/A
24
Q

What are transcription factors?

A

Proteins that regulate transcription by binding specific sequences in or near the promoter region
- Helix–turn–helix motif

25
Q

What are the mechanisms of action of transcription factors?

A
  1. Bind RNAP and recruit to promoter (usual mechanism)
    - Ex. CRP
  2. Bind and alter DNA structure
    - Ex. IHF
26
Q

Give an example of transcription regulation.

A

Ex. lac operon
1. Lactose induces production of genes of the lac operon
- β-galactosidase
- Permease
- Transacetylase
2. Repressor protein produced from lacI gene with its own promoter

27
Q

What are negative regulators?

A

Aka repressors
- Bind to nucleotide sequences in the DNA called operator regions and inhibit transcription
- Ex. lac repressor binds to the operator region that overlaps the start site of the first gene in the lac operon

28
Q

What are positive regulators?

A

Bind to the promoter region or to enhancer sites, which are sequences upstream from the promoter region
- May make contact with RNA polymerase and promote its binding to the promoter region
- Some may facilitate the “melting” of DNA to form the transcription bubble

29
Q

Draw a typical gene structure found in bacteria.

A
30
Q

List 3 proteins that regulate transcription.

A
  • RNAP
  • Sigma factors
  • Repressor protein
31
Q

What is translation?

A

Protein synthesis

32
Q

How does prokaryotic translation differ from eukaryotic translation?

A
  • Different ribosomal subunits (50S and 30S)
  • Different initiating tRNA (formyl methionine)
  • Only 3 initiation factors
33
Q

What are the 3 stages of protein synthesis?

A
  1. Initiation
  2. Elongation
  3. Termination
34
Q

What is the pre-initation complex formed with (protein synthesis)?

A
  • 30S ribosomal subunit
  • mRNA
  • fMet-tRNA
  • Initiation factors (IF-1, IF-2, IF-3)
35
Q

What is the initiation complex formed with (protein synthesis)?

A
  • 50S subunit added –> 70S ribosome
  • Shine–Dalgarno sequence positions the mRNA on the ribosome
36
Q

What is the Shine-Dalgarno sequence?

A

String of about 5 to 10 nucleotides approximately 6 to 10 nucleotides upstream of the start codon

37
Q

What is the function of the Shine-Dalgarno sequence?

A

Positions the mRNA on the ribosome

38
Q

Outline the steps of elongation (protein synthesis).

A
  1. Begins at an AUG codon (start codon) –> codes for methionine in the initiator region of the mRNA
    a. Leader sequence: untranslated region at the 5′ end of the mRNA (if RNA transcription begins upstream of start codon)
  2. Chain elongation is initiated when an incoming aminoacylated tRNA enters the A site on the ribosome
  3. GTP hydrolysis occurs when correct codon-anticodon pairing is achieved –> EF-Tu-GDP is released from the ribosome
  4. GDP dissociates to facilitate recharging with GTP
  5. Peptide bond forms when the free α-amino group on the amino acid bound to the tRNA in the A site displaces the tRNAf in the P site
  6. Translocation: EF-G/GTP induces a conformational change, moving the mRNA by one codon length, facilitating the movement of tRNAs, and opening the A site for the next charged tRNA, ensuring the progression of the ribosome along the mRNA during protein synthesis
39
Q

Outline the steps of termination (protein synthesis).

A
  1. Chain termination is initiated when a stop codon (UAA, UGA, or UAG) reaches the A site
  2. Release factors (proteins) bind to the stop codons and cause the release of the completed polypeptide from the ribosome
  3. Binding of a release factor (RF-1 or RF-2) occurs in the A site and allows water access to the peptidyl transferase center
  4. Peptide is hydrolyzed from the terminal tRNA
  5. Ribosome is recycled by the action of two other proteins: RRF (ribosome recycling factor) and EF-G/GTP
  6. Ribosome is disassembled by GTP hydrolysis in the presence
    of RRF
40
Q

Why are transcription and translation coupled in bacteria?

A

No organelles separating processes in prokaryotes

41
Q

How does coupled transcription and translation occur?

A
  1. Ribosomes attach to 5′ end of mRNA before the message is completely transcribed
  2. Move along mRNA toward the 3′ end as transcription continues
42
Q

What are polysomes?

A

Several ribosomes attached to single mRNA –> efficiency (increases the rate of protein synthesis)

43
Q

What is attenuation?

A

Regulation after initiation
- Only found in prokaryotes
- Requires coupling of transcription and translation
- Based on mRNA structure
- mRNA forms hairpin structures: terminator and anti-terminator hairpins

44
Q

Give an example of attenuation.

A

Ex. Tryptophan synthesis
1. Leader sequences of mRNA transcribed from trp operon contains:
- Codons for tryptophan
- 4 segments that can form hairpin structures
2. Occurs when all amino acids are low in concentration
- Translation not initiated
3. When Trp adequate
- Hairpin structure formed between 3rd and 4th segments functions as terminator –> transcription stops
4. When Trp limited
- 2nd and 3rd segments form hairpin structure that prevents the formation of the terminator hairpin (anti-terminator) –> transcription continues

45
Q

What antibiotics inhibit protein synthesis?

A
  • Chloramphenicol
  • Erythromycin
  • Streptomycin
  • Tetracyclin

Bacterial Physiology (BIOL 471) (15 decks)

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