Ch. 11 Flashcards
What fundamental principles does the synthesis of macromolecules follow?
- Modular: made up of repeating polymers
- Dehydration reactions are involved in polymerization
- Reactions are energetically unfavorable
What is transcription?
RNA synthesis
What are the 3 stages of RNA synthesis?
- Initiation
- Elongation
- Termination
What are the requirements for the initiation of RNA synthesis?
- Promoter
- RNA polymerase
- Sigma factors
What is the promoter?
DNA region that RNA polymerase binds to
What is the function of the Pribnow box?
- Aka the -10 sequence
- It is what RNA polymerase recognizes to bind to the promoter
What is the function of RNA polymerase?
Transcribing RNA
What is the function of sigma (σ) factors?
Responsible for the recognition of promoters by RNA polymerase (bind to RNA polymerase to form the RNAP holoenzyme)
At what point during RNA synthesis does elongation occur?
After about 12 NTPs have been added
What happens during the initiation stage of RNA synthesis?
- Formation of the closed complex
- RNAP holoenzyme bound to promoter region
- DNA enters but remains dsDNA
- Reversible - Formation of open complex
- DNA duplex unwinds at promoter region to form open complex
- Irreversible - Binding of initiating ribonucleotides
- Transcription is usually initiated with ATP or GTP
- NTPs enter active site through a 2º channel
What happens during the elongation stage of RNA synthesis?
- 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)
What are the 2 patterns of termination of RNA synthesis?
- Factor-independent
- Rho-dependent
Explain factor-independent termination.
- Self-complementary sequence of bases
- RNA hybridizes itself (hairpin loop formation)
- RNA spontaneously dissociates from DNA
- RNAP dissociates
Explain Rho-dependent termination.
- 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
What methods do bacteria use to regulate protein synthesis at the transcriptional level?
- Sigma factors/anti-sigma factors
- Operons
- Transcription factors
Transcription is commonly regulated at the initiation step by proteins that affect _____, _____, and _____.
- the binding of RNAP to the promoter
- the “melting” of DNA to form transcription bubble
- movement of RNAP along DNA (elongation)
Selection of promoters by RNA polymerase is dependent on the _____ that associates with it.
Sigma factor
What are the 2 families of sigma factors?
- σ-70
- σ-54
What do anti-sigma factors do?
Bind sigma factors and inhibit their activity
Give an example of an anti-sigma factor (what it is, how it works, what happens to it)?
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
What is an operon?
A sequence of DNA containing a cluster of genes under the control of a single promoter
- Polycistronic mRNA
What are the 3 basic components of an operon?
- Promoter
- Operator
- Genes
What binds to each of the components of an operon?
- Promoter: RNAP binds
- Operator: repressor protein binds
- Genes: N/A
What are transcription factors?
Proteins that regulate transcription by binding specific sequences in or near the promoter region
- Helix–turn–helix motif
What are the mechanisms of action of transcription factors?
- Bind RNAP and recruit to promoter (usual mechanism)
- Ex. CRP - Bind and alter DNA structure
- Ex. IHF
Give an example of transcription regulation.
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
What are negative regulators?
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
What are positive regulators?
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
Draw a typical gene structure found in bacteria.
List 3 proteins that regulate transcription.
- RNAP
- Sigma factors
- Repressor protein
What is translation?
Protein synthesis
How does prokaryotic translation differ from eukaryotic translation?
- Different ribosomal subunits (50S and 30S)
- Different initiating tRNA (formyl methionine)
- Only 3 initiation factors
What are the 3 stages of protein synthesis?
- Initiation
- Elongation
- Termination
What is the pre-initation complex formed with (protein synthesis)?
- 30S ribosomal subunit
- mRNA
- fMet-tRNA
- Initiation factors (IF-1, IF-2, IF-3)
What is the initiation complex formed with (protein synthesis)?
- 50S subunit added –> 70S ribosome
- Shine–Dalgarno sequence positions the mRNA on the ribosome
What is the Shine-Dalgarno sequence?
String of about 5 to 10 nucleotides approximately 6 to 10 nucleotides upstream of the start codon
What is the function of the Shine-Dalgarno sequence?
Positions the mRNA on the ribosome
Outline the steps of elongation (protein synthesis).
- 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) - Chain elongation is initiated when an incoming aminoacylated tRNA enters the A site on the ribosome
- GTP hydrolysis occurs when correct codon-anticodon pairing is achieved –> EF-Tu-GDP is released from the ribosome
- GDP dissociates to facilitate recharging with GTP
- 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
- 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
Outline the steps of termination (protein synthesis).
- Chain termination is initiated when a stop codon (UAA, UGA, or UAG) reaches the A site
- Release factors (proteins) bind to the stop codons and cause the release of the completed polypeptide from the ribosome
- Binding of a release factor (RF-1 or RF-2) occurs in the A site and allows water access to the peptidyl transferase center
- Peptide is hydrolyzed from the terminal tRNA
- Ribosome is recycled by the action of two other proteins: RRF (ribosome recycling factor) and EF-G/GTP
- Ribosome is disassembled by GTP hydrolysis in the presence
of RRF
Why are transcription and translation coupled in bacteria?
No organelles separating processes in prokaryotes
How does coupled transcription and translation occur?
- Ribosomes attach to 5′ end of mRNA before the message is completely transcribed
- Move along mRNA toward the 3′ end as transcription continues
What are polysomes?
Several ribosomes attached to single mRNA –> efficiency (increases the rate of protein synthesis)
What is attenuation?
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
Give an example of attenuation.
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
What antibiotics inhibit protein synthesis?
- Chloramphenicol
- Erythromycin
- Streptomycin
- Tetracyclin
