Lecture 8 - gene expression Flashcards
General steps of protein synthesis from DNA
- RNA Pol binds promoter sequence on coding strand
- Transcription of the template strand –> mRNA; untranslated leader and trailer sequences flank the coding sequence
- Translation of mRNA from the start codon to the stop codon
Temporal relationship between transcription and translation in bacteria
Transcription and translation are coupled (occur simultaneously)
Transcription
Process by which RNA copy of DNA template is synthesized by RNA polymerase
Translation
Process by which mRNA is converted into a specific peptide sequence
RNA polymerase
Multi-subunit enzyme that copies DNA templates into ssRNA molecules called transcripts
How many types of RNA polymerase (holoenzyme) are present in bacteria?
1
Composition of RNA pol in bacteria
- core polymerase: (alpha2, beta, beta prime, and omega) minimum assembly required for RNA elongation
- sigma factor: required to identify promoter elements
How does sigma factor identify the promoter region?
Scans the DNA for consensus sequences at -35 and -10 positions relative to the transcription start site (+1)
Where is the active site of RNA polymerase?
In the “clam shell” structure made by beta and beta prime subunits
What is sigma 70 in E. coli?
“housekeeping” sigma factor that is responsible for general transcription
Why do bacteria contain several different sigma factors?
To control gene expression at the transcription level
What is a strong promoter?
A promoter sequence that binds RNA pol well to allow for multiple rounds of successive transcription
Topological organization of -35 and -10 promoter regions?
Presented on the same side of the DNA to aid in promoter efficiency
General steps of transcription initiation
- RNA pol holoenzyme (with sigma factor) binds promoter to form closed complex
- -10 region of DNA spontaneously unwinds to form open complex
- Sigma factor is released
Closed complex vs open complex
Closed: RNA pol bound to dsDNA
Open: RNA pol bound to DNA after strand separation
How many base pairs in the heteroduplex inside RNA pol during elongation?
7-9 base pairs
How do nucleotides get into the active site of RNA pol?
NTP channel (nucleotide triphosphate) allows nucleotides to pass into the interior of the protein to the active site
Types of transcription termination
Rho dependent and rho independent (aka “intrinsic” termination)
Requirements of rho dependent transcription termination
Relies on Rho, and ATPase, that interacts with rut sites (rho utilization sites) at the 3’ end of the gene
Requirements of rho independent transcription termination
Requires a CG rich stem-loop in the nascent transcript followed by 4-8 consecutive U residues
How does rho dependent transcription termination work?
Rho binds around rut sites on nascent mRNA and pulls RNA from RNA polymerase
How does rho independent transcription termination work?
Stem-loop secondary structure forms after mRNA exits the active site, multiple RNA uracil-DNA adenine bonds in the active site.
- ribo U to deoxy A is a very unstable bond that causes release of the mRNA
Steps of translation initiation
- Small ribosome subunit (16S rRNA) interacts with ribosome binding site on mRNA; initiation factors (IFs) regulate this process
- Initiation factor interacts with initiator tRNA at start codon
- Large ribosome subunit is recruited
Steps of translation elongation
- Ef-Tu-GTP binds tRNA and guides tRNA to the A site of the ribosome
- Peptide bond formed between nascent peptide strand and new peptide
- Ribosome moves one codon down the mRNA
Components of EF-Tu-GTP
EF: elongation factor
Tu
GTP
Steps of translation termination
- Stop codon enters A site
- No tRNA for stop codon; protein release factor binds A site
- Release and recycling factors (RFs) disassemble ribosomal subunits
How does streptomycin and other aminoglycosides interfere with protein synthesis
Interferes with mRNA decoding.
- positively charged, binds rRNA
- binds decoding center and induces translational errors
- allows ribosome to accept incorrect tRNAs
How does tetracycline interfere with protein synthesis
Inhibits binding of aminoacyl-tRNA to A site (blocks tRNA delivery)
How does chloramphenicol interfere with protein synthesis
Blocks peptide bond formation
How does puromycin interfere with protein synthesis
Prematurely releases nascent peptides from P-site tRNA –> premature termination
How does erythromycin and other macrolides interfere with protein synthesis
Blocks elongation and induces “drop off” of peptidyl-tRNA from ribosome
How does fusidic acid interfere with protein synthesis
Prevents translocation by stopping activity of EFs
Downside of streptomycin and chloramphenicol
Can accidentally attack mitochondrial ribosomes in eukaryotes
Downside of antibiotics that interfere with protein synthesis
Many have evolved in bacteria to kill other bacteria –> many bacteria have innate resistance
