Lecture 8 - gene expression

Question 1

General steps of protein synthesis from DNA

Answer 1

1. RNA Pol binds promoter sequence on coding strand
2. Transcription of the template strand –> mRNA; untranslated leader and trailer sequences flank the coding sequence
3. Translation of mRNA from the start codon to the stop codon

Question 2

Temporal relationship between transcription and translation in bacteria

Answer 2

Transcription and translation are coupled (occur simultaneously)

Question 3

Transcription

Answer 3

Process by which RNA copy of DNA template is synthesized by RNA polymerase

Question 4

Translation

Answer 4

Process by which mRNA is converted into a specific peptide sequence

Question 5

RNA polymerase

Answer 5

Multi-subunit enzyme that copies DNA templates into ssRNA molecules called transcripts

Question 6

How many types of RNA polymerase (holoenzyme) are present in bacteria?

Answer 6

1

Question 7

Composition of RNA pol in bacteria

Answer 7

• core polymerase: (alpha2, beta, beta prime, and omega) minimum assembly required for RNA elongation
• sigma factor: required to identify promoter elements

Question 8

How does sigma factor identify the promoter region?

Answer 8

Scans the DNA for consensus sequences at -35 and -10 positions relative to the transcription start site (+1)

Question 9

Where is the active site of RNA polymerase?

Answer 9

In the “clam shell” structure made by beta and beta prime subunits

Question 10

What is sigma 70 in E. coli?

Answer 10

“housekeeping” sigma factor that is responsible for general transcription

Question 11

Why do bacteria contain several different sigma factors?

Answer 11

To control gene expression at the transcription level

Question 12

What is a strong promoter?

Answer 12

A promoter sequence that binds RNA pol well to allow for multiple rounds of successive transcription

Question 13

Topological organization of -35 and -10 promoter regions?

Answer 13

Presented on the same side of the DNA to aid in promoter efficiency

Question 14

General steps of transcription initiation

Answer 14

1. RNA pol holoenzyme (with sigma factor) binds promoter to form closed complex
2. -10 region of DNA spontaneously unwinds to form open complex
3. Sigma factor is released

Question 15

Closed complex vs open complex

Answer 15

Closed: RNA pol bound to dsDNA
Open: RNA pol bound to DNA after strand separation

Question 16

How many base pairs in the heteroduplex inside RNA pol during elongation?

Answer 16

7-9 base pairs

Question 17

How do nucleotides get into the active site of RNA pol?

Answer 17

NTP channel (nucleotide triphosphate) allows nucleotides to pass into the interior of the protein to the active site

Question 18

Types of transcription termination

Answer 18

Rho dependent and rho independent (aka “intrinsic” termination)

Question 19

Requirements of rho dependent transcription termination

Answer 19

Relies on Rho, and ATPase, that interacts with rut sites (rho utilization sites) at the 3’ end of the gene

Question 20

Requirements of rho independent transcription termination

Answer 20

Requires a CG rich stem-loop in the nascent transcript followed by 4-8 consecutive U residues

Question 21

How does rho dependent transcription termination work?

Answer 21

Rho binds around rut sites on nascent mRNA and pulls RNA from RNA polymerase

Question 22

How does rho independent transcription termination work?

Answer 22

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

Question 23

Steps of translation initiation

Answer 23

1. Small ribosome subunit (16S rRNA) interacts with ribosome binding site on mRNA; initiation factors (IFs) regulate this process
2. Initiation factor interacts with initiator tRNA at start codon
3. Large ribosome subunit is recruited

Question 24

Steps of translation elongation

Answer 24

1. Ef-Tu-GTP binds tRNA and guides tRNA to the A site of the ribosome
2. Peptide bond formed between nascent peptide strand and new peptide
3. Ribosome moves one codon down the mRNA

Question 25

Components of EF-Tu-GTP

Answer 25

EF: elongation factor
Tu
GTP

Question 26

Steps of translation termination

Answer 26

1. Stop codon enters A site
2. No tRNA for stop codon; protein release factor binds A site
3. Release and recycling factors (RFs) disassemble ribosomal subunits

Question 27

How does streptomycin and other aminoglycosides interfere with protein synthesis

Answer 27

Interferes with mRNA decoding.
- positively charged, binds rRNA
- binds decoding center and induces translational errors
- allows ribosome to accept incorrect tRNAs

Question 28

How does tetracycline interfere with protein synthesis

Answer 28

Inhibits binding of aminoacyl-tRNA to A site (blocks tRNA delivery)

Question 29

How does chloramphenicol interfere with protein synthesis

Answer 29

Blocks peptide bond formation

Question 30

How does puromycin interfere with protein synthesis

Answer 30

Prematurely releases nascent peptides from P-site tRNA –> premature termination

Question 31

How does erythromycin and other macrolides interfere with protein synthesis

Answer 31

Blocks elongation and induces “drop off” of peptidyl-tRNA from ribosome

Question 32

How does fusidic acid interfere with protein synthesis

Answer 32

Prevents translocation by stopping activity of EFs

Question 33

Downside of streptomycin and chloramphenicol

Answer 33

Can accidentally attack mitochondrial ribosomes in eukaryotes

Question 34

Downside of antibiotics that interfere with protein synthesis

Answer 34

Many have evolved in bacteria to kill other bacteria –> many bacteria have innate resistance