Chapter 8

Question 1

central dogma

Answer 1

dna-mrna-protein

Question 2

Transcription

Answer 2

copying DNA to RNA

RNA polymerase

Question 3

translation

Answer 3

decoding RNA to synthesize protein

Ribosome

Question 4

Core polymerase =

Answer 4

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

Question 5

Sigma factors

Answer 5

guide RNA polymerase to beginn. of genes-to promoter

Question 6

in e coli the sigma factor is

Answer 6

“housekeeping” sigma factor is RpoD s^70 (s=sigma symbol)

Question 7

promoter

Answer 7

• +1 nt

* promoter sequence generally (depends on sigma factor) at -10 and -35 nt

Question 8

transcription begins

Answer 8

• Typically with a purine (A, G)

* Sigma factor releases after first few bases are added (~9)

Question 9

Transcription Elongation

Answer 9

• Core RNAP synthesizes RNA ~45 bases per sec
• Forms 17-bp transcription bubble
• Energy for base addition comes release of pyrophosphate

Question 10

Transcription Termination

• Rho-dependent termination

Answer 10

• 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

Question 11

Transcription Termination continued

• Rho-independent termination

Answer 11

• 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

Question 12

Rifamycin B binds to

Answer 12

b’ subunit near the Mg2+ active site and blocks the RNA exit channel. (antibiotics block transcription)

Question 13

codons

Answer 13

nucleotides triplets that correspond to amino acids

Question 14

Translation – The Ribosome

Answer 14

• Small subunit (30S) + large subunit (50S) = ribosome
(70S)
30S= 21 ribosomal proteins + 16S rRNA
50S=31 ribosomal proteins+ 5S rRNA + 23S rRNA

Question 15

23S rRNA

Answer 15

ribozyme activity-peptidyltransferase

Question 16

The ribosome has three binding sites for tRNA

Answer 16

E,P,A

Question 17

Initiation requires three initiation factors (IF)

Answer 17

IF1, IF2, IF3

Question 18

Translation – Elongation
1.
2.
3.

Answer 18

• 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

Question 19

Translation – Termination
stops 
release
peptidyltransferase
rf3 enters
rrf
gtp hydrolysis
if3 binds

Answer 19

• 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

Question 20

Streptomycin

Answer 20

binds to 30S subunit – interferes with codon-anticodon recognition site –results in mistranslated protein sequences

Question 21

Tetracycline

Answer 21

binds to 30S subunit and blocks aminoacyl-tRNA binding to the A site

Question 22

Chloramphenicol

Answer 22

inhibits peptidyltransferase activity of the 23S rRNA

Question 23

Erythromycin

Answer 23

binds to 23S rRNA and interferes with translocation

Question 24

Protein structure may be modified after translation

Answer 24

• N-formyl group may be removed by methionine deformylase (leaves a regular methionine)
•entire methionine may be removed by methionyl
aminopeptidase

Question 25

degrons

Answer 25

Proteins contain degradation signals

Question 26

N-terminal rule (protein degradation)

Answer 26

* 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

Question 27

Abnormally folded proteins are

Answer 27

recognized by proteases bc hydrophobic regions are exposed - Progressively degraded into smaller and smaller pieces - involve ATP-dependent endoproteases like Lon or ClpP

Question 28

cell envelope contains | How do they get to the specific locations?

Answer 28

proteins * Some proteins are secreted completely out of cell * Require special export systems

Question 29

Proteins meant for the inner membrane are

Answer 29

tagged with hydrophobic N-terminal signal sequences of 15-30 AA Bound by the signal recognition particle (SRP)

Question 30

signal recognition particle (SRP)

Answer 30

pauses translation until its delivered to membrane

Question 31

Journeys through the outer membrane

Answer 31

* Export out of the cell | * 7 secretion systems – classified based on their structure

Question 32

How many GTP molecules would it take to synthesize a | protein 100 amino acids in length?

Answer 32

``` there are 99 bonds. GTPs 1-initiation 2-bond 1-termination you do 99x2+1+1=200 ```

Question 33

rna polymerase holoenzyme

Answer 33

core polymerase + sigma factor

Question 34

protein export to periplasm: general secretion pathway

Answer 34

* 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