Chapter 8 Flashcards
central dogma
dna-mrna-protein
Transcription
copying DNA to RNA
RNA polymerase
translation
decoding RNA to synthesize protein
Ribosome
Core polymerase =
2 alpha (a) subunits + 1 beta (b) subunit + 1 beta-prime (b’) subunit + [1 omega (w) subunit (not required for transcription)] B' subunit: houses Mg2+catalytic site
Sigma factors
guide RNA polymerase to beginn. of genes-to promoter
in e coli the sigma factor is
“housekeeping” sigma factor is RpoD s^70 (s=sigma symbol)
promoter
- +1 nt
* promoter sequence generally (depends on sigma factor) at -10 and -35 nt
transcription begins
- Typically with a purine (A, G)
* Sigma factor releases after first few bases are added (~9)
Transcription Elongation
- Core RNAP synthesizes RNA ~45 bases per sec
- Forms 17-bp transcription bubble
- Energy for base addition comes release of pyrophosphate
Transcription Termination
• Rho-dependent termination
- Rho – hexameric protein that binds to C-rich sequences in the open reading frame (ORF)
- ATPase activity enables rho to move 5’ to 3’
- RNAP pauses at termination site
- Rho reaches RNAP and unwinds the RNA-DNA, releasing RNA and RNAP
Transcription Termination continued
• Rho-independent termination
- Intrinsic termination
- hairpin loop with GC-rich stem followed by poly-U
- RNAP pauses at poly-U
- U-A RNA-DNA hybrid is unstable
- hairpin causes RNAP to leave DNA
- NusA assists in RNAP pausing and hairpin formation
Rifamycin B binds to
b’ subunit near the Mg2+ active site and blocks the RNA exit channel. (antibiotics block transcription)
codons
nucleotides triplets that correspond to amino acids
Translation – The Ribosome
• Small subunit (30S) + large subunit (50S) = ribosome
(70S)
30S= 21 ribosomal proteins + 16S rRNA
50S=31 ribosomal proteins+ 5S rRNA + 23S rRNA
23S rRNA
ribozyme activity-peptidyltransferase
The ribosome has three binding sites for tRNA
E,P,A
Initiation requires three initiation factors (IF)
IF1, IF2, IF3
Translation – Elongation
1.
2.
3.
• Elongation factors (EF-Tu, EF-G) are complexed to GTP, which is used for energy
• 16 AA/sec
1. Aminoacyl-tRNA binds to the A site
• Bound to EF-Tu-GTP
2. Peptide bond forms between the new amino acid and growing peptide in position P
• 23S peptidyltransferase
3. Translocation - movement to the next codon
• EF-G-GTP
Translation – Termination stops release peptidyltransferase rf3 enters rrf gtp hydrolysis if3 binds
- Stop codon moves into A site
- Release factor RF1 or RF2 enters A site
- Peptidyltransferase activity releases the peptide from tRNA in the P site
- RF3 enters and triggers release of RF1 or RF2
- Ribosome recycling factor (RRF) enters A site w/ EF-G GTP
- GTP hydrolysis undocks the ribosomal subunits
- IF3 binds 30S, preventing 50S from re-docking
Streptomycin
binds to 30S subunit – interferes with codon-anticodon recognition site –results in mistranslated protein sequences
Tetracycline
binds to 30S subunit and blocks aminoacyl-tRNA binding to the A site
Chloramphenicol
inhibits peptidyltransferase activity of the 23S rRNA
Erythromycin
binds to 23S rRNA and interferes with translocation
Protein structure may be modified after translation
• N-formyl group may be removed by methionine deformylase (leaves a regular methionine)
•entire methionine may be removed by methionyl
aminopeptidase
degrons
Proteins contain degradation signals
N-terminal rule (protein degradation)
- Leu, Phe, Trp, Tyr – short, 2 minute half-life
- Asp, Glu, Cys – longer half-life
- ClpS recognizes the N-terminal AA and presents protein to ClpAP protease
Abnormally folded proteins are
recognized by proteases bc hydrophobic regions are exposed
- Progressively degraded into smaller and smaller pieces
- involve ATP-dependent endoproteases like Lon or ClpP
cell envelope contains
How do they get to the specific locations?
proteins
- Some proteins are secreted completely out of cell
- Require special export systems
Proteins meant for the inner membrane are
tagged with hydrophobic N-terminal signal sequences of 15-30 AA
Bound by the signal recognition particle (SRP)
signal recognition particle (SRP)
pauses translation until its delivered to membrane
Journeys through the outer membrane
- Export out of the cell
* 7 secretion systems – classified based on their structure
How many GTP molecules would it take to synthesize a
protein 100 amino acids in length?
there are 99 bonds. GTPs 1-initiation 2-bond 1-termination you do 99x2+1+1=200
rna polymerase holoenzyme
core polymerase + sigma factor
protein export to periplasm: general secretion pathway
- Peptide completely translated in cytoplasm
- protein wraps around SecB
- SecB delivers the protein to SecA and SecYEG
- SecA inserts the protein in SecYEG using ATP
- LepB cleaves protein
- protein must fold in periplasm
