Prokaryotic Gene Expression

Question 1

what are the proteins in a prokaryotic RNA pol?

Answer 1

beta, beta’, alpha 1 and 2, and omega–> sigma is added for specificity, but the pol will work without it

Question 2

RNA pol in proks start to transcribe from NICKED DNA

Answer 2

ya

Question 3

RNApol has 5’ to 3’ pol and helicase activity

Answer 3

ya

Question 4

what is the purpose of the omega unit?

Answer 4

helps assembly

Question 5

what are the alpha units required for?

Answer 5

required for assembly, have some DNA specificity

Question 6

what are the beta subunits required for?

Answer 6

catalytic proteins for RNA polymerization

Question 7

assembly order for RNA pol

Answer 7

alpha dimerizes, binds beta, omega and beta’ bind, then both complexes bind together–sigma comes in later after binding to DNA

Question 8

different sigma factors for different genes promoters–> sigma 70 is common for many genes though

Answer 8

ya

Question 9

sigma has 4 conserved domains–> domains 2 and 4 contact the DNA t the positions -10 and -35, respectively

Answer 9

ya

Question 10

the closer the consensus seq is for sigma tobind to, the more increased the probablility is that RNA pol will bind there

Answer 10

ya

Question 11

-10 box is the Pribnow box–TATA boxes are eukaryotic

Answer 11

aya

Question 12

additional binding sites for RNA pol

Answer 12

-40-60, UP element and extended -10, TG seq at -14/-15

Question 13

what are the purpose of Gre A and B

Answer 13

enhance hydrolytic removal–>RNA pol backtracks 3-10 nt and cuts by endonuclease activity–involved in proofreading of RNA pol

Question 14

what are the proofreading activities of RNA?

Answer 14

pryphosphorolytic rxn–cuts two phosphates off a triphosphate nt; hydrolytic removal–>RNA pol backtracks 3-10 bases and cuts via endonuc activity

Question 15

termination in bacteria

Answer 15

2 types: rho depedent and rho independent

Question 16

what is rho independent termination?

Answer 16

utlized GC-rich inverted repeat seq; has no effect on DNA but causes a stem-loop form from teh RNA; stem forms and yanks RNA rom the pol; need a stretch of ~3 A:U pairs so binding will be weak enough to free the RNA

Question 17

what is rho dependent termination?

Answer 17

used less often that rho independent; rho is a hexameric protein that binds the RNA t the rut site (rho utilization); rho binds RNA & yanks it out; • Rho-dependent might help to make it more error proof
Rut site can only be bound after translational stop site–otherwise ribosomes would constantly be feeding on this and disrupting Rho from reaching the site

Question 18

the rut site is high in Cs

Answer 18

ya

Question 19

translation starts before transcription is finished

Answer 19

ya

Question 20

what are polycistronic genes?

Answer 20

several genes are transcribed to one mRNA, but one mRNA makes many proteins–> generally, the expression of several genes at once is useful because they will normally be needed to be used together

Question 21

describe the 70S ribosome

Answer 21

2 subunits: 50S and 30S; 50S contains the 23 and 5 S and 34 proteins; 30S contains the 16 S rRNA and 21 protiens

Question 22

what role does the 16S rRNA play?

Answer 22

ensures accurate tRNA binding and placement of translational initiation (through hoogsteen binding)

Question 23

what role does the 23S rRNA play?

Answer 23

produces peptide bonds between amino acids and the growing protein

Question 24

what role does the 5S rRNA play?

Answer 24

structural integrity

Question 25

what are the initiation factors and what do they do?

Answer 25

IF-3 binds to the 30S subunit and prevent association with the 50s for the time being; IF 1+2 come in and bind the 30S (IF 2 has fMet on it) at the S-D seq; IF 1+3 are lost so now the 50S subunit can bind–> fMet now in P site; IF2 is released from the A site–>free A-site can now bind a tRNA

Question 26

describe synthesis of a protein

Answer 26

the 23S rRNA catalyzes the transfer of the growing polypeptide to the newest aa; ribosome slides forward one codon–>newest tRNA enters P site from A site, and the tRNA in the P site goes to teh E site–>now a new tRNA can enter the P site

Question 27

describe elongation in proks

Answer 27

aminoacyl-tRNA in brought to A site by EF-Tu in a GTP-hydrolyzing step; 23S ribosomal RNA transfers the growing peptide to teh N-terminus of the new aa; EF-G ratchets the transition-state ribo forward so that A-site tRNA with the protein in placed into the P ist

Question 28

speed of elongation?

Answer 28

60 nt/sec or 20 aa/sec

Question 29

describe translation termination in proks

Answer 29

no tRNA binds to a stop codon; instead release factors 1 or 2 (depending on the stop codon) binds to the stop codon and 23S rRNA hydrolyzes the bond bw tRNA and protein; RF-3 then dissembles the ribosome