Lecture 45- Prokaryotic Transcription & Gene Control

Question 1

What are trans-acting factors?

Examples Include:

Answer 1

diffusible and thus can function at multiple sites in genome

DNA binding protein such as transcription factors, some regulatory RNAs

Question 2

What are cis acting elements?

Examples Include:

Answer 2

tied closely to the gene

DNA sequence elements such as promoters and binding sites for trans-acting factors

Question 3

What is RNA and what are it’s 4 defining characteristics?

Answer 3

linear single-stranded polynucleotide chain with:

• sequence always read 5’–>3’
• ribose-phosphate backbond
• U instead of T
• intramolecular base pairing that yields complex secondary and tertiary structure

Question 4

Define messenger RNA (mRNA)

Answer 4

RNA copy of coding strand of DNA from bacterial gene. Carries base sequence encoding amino acid sequence for a protein.

Question 5

Define transfer RNA (tRNA)

Answer 5

RNA molecule that carries AA into catalytic site of ribosome. tRNA base pairs to mRNA to ensure selection of correct AA to be incorporated into nascent polypeptide chain

Question 6

Define ribosomal RNA (rRNA)

Answer 6

Structural components of ribosome, enzyme that catalyzes protein synthesis

Question 7

What is a transcription unit and the process of transcription?

Answer 7

DNA that can be transcribed into a single RNA molecule catalyed by RNA polymerase

Question 8

Where does a transcription unit start and end?

What is the finished molecule?

Answer 8

stars at promoter, ends at terminator

Primary transcript

Question 9

During mRNA synthesis, what is considered upstream and downstream?

Answer 9

Upstream: closer to the 5’ end

Downstream: closer to the 3’ end

Question 10

What gives the location of the promoter and what is its value?

Answer 10

Transcription start site, +1

Question 11

What term defines close to the promoter?

Far from the promoter?

Answer 11

Proximal

Distal

Question 12

The RNA __ sequence is similar to the DNA coding strand

Answer 12

The RNA TRANSCRIPT sequence is similar to the DNA coding strand

Question 13

Original 2 stranded DNA consists of?

Answer 13

Coding and template strand

Question 14

When DNA gets unwound, we use the ____ strand to transcribe. The ____ that is formed is the compliment of the ____ strand.

Answer 14

When DNA gets unwound, we use the template strand to transcribe. The transcript that is formed is the compliment of the template strand.

Question 15

How are coding and template strands defined?

Answer 15

For each individual gene

Question 16

What are operons?

Answer 16

coordinately regulated gene clusters, cluster of bacterial genes

Question 17

___ are arranged in 5’ –> 3’ in transcription unit

Answer 17

OPEN READING FRAMES are arranged in 5’ –> 3’ in transcription unit

Question 18

Use of one promoter and terminator yields a ____

Answer 18

Use of one promoter and terminator yields a POLYCISTRONIC mRNA

Question 19

How many proteins does the atp operon encode?

Answer 19

9

Question 20

A ___ is a cis-acting element in a genome where ___ ______ binds to initiate transcription

Answer 20

A PROMOTER is a cis-acting element in a genome where RNA POLYMERASE binds to initiate transcription

Question 21

Where are bacterial promoters’ defined sequence elements?

Answer 21

• the -35 region
• the -10 region

Question 22

Where is the spacer in prokaryotic promoters?

Answer 22

between -35 and -10 regions

Question 23

What do most RNA transcripts start with?

Answer 23

Purine (+1) within the sequence CAT

Question 24

What is the bacterial consenus promoter?

Answer 24

-35 Spacer -10 Spacer +1

TTGACA– 16-18bp – TATAAT –5-9bp - CAT

Question 25

What are constitutive promoters?

Answer 25

Actively initiate transcription continuously for ‘housekeeping genes’

Question 26

What does the constitutive promoter strength depend on?

Answer 26

sequence

Question 27

What is a major determinant of the rate of transcription initiation for constitutive promoter?

Answer 27

Similarity to consensus sequence

Question 28

The closer to the consensus sequence the promoter is, the faster or slower the rate of transcription?

Answer 28

faster

Question 29

How many polymerases does E.coli have that catalyzes transcription in 5’ –>3’. What is it (they) called?

Answer 29

1, but 2 types

RNA polymerase (465kD)

Question 30

What does RNA polymerase holoenzyme do?

Answer 30

Responsible for transcription initiation and synthesis of first 10 nucleotides of RNA chain

Question 31

What are the subunits of the holoenzyme?

Answer 31

alpha₂, beta, beta’, omega, sigma,

• sigma recognizes promoter
• alpha₂ (40kd) essentail for enzyme assembly involved in interaction with activators
• beta (155kD) and beta’ (160kD) form catalytic core
• omega provides structural stability

Question 32

What does RNA polymerase core enzyme consist of and do?

Answer 32

alpha₂, beta, beta’, omega

carries out transcription elongation

Question 33

What holoenzyme binds the most promoters in the E.coli genome and what is its function?

Answer 33

sigma 70! (70% are sigma70)

housekeeping

Question 34

What does RNA polymerase require (3)?

Answer 34

• DNA template
• no primer
• substrates that are ribonucleotide triphosphates

Question 35

T/F: RNA polymerases have proof-reading exonuclease activity.

Why/why not?

Answer 35

FALSE. RNA polymerases DO NOT have proof-reading exonuclease activity. (Error rate: 10^-4 to 10^-5)

It can afford it bc defective mRNA –> protein which will undergo turnover quickly. Whereas, if DNA makes a mistake, mutation in the genome could kill the cell

Question 36

Transcription Initiation

1. __ subunit binds to the ______
2. Rest of the RNA pol binds to the ____ subunit which produces a _______ that binds to the promoter and creates a “_____” complex
3. Unwinding of DNA forms a ______ _____, #-#bp, to produce an “___” complex
4. ________ initiates RNA synthesis for ~__ nt

Answer 36

Transcription Initiation

1. Sigma subunit binds to the promoter
2. Rest of the RNA pol binds to the Sigma subunit which produces a holoenzyme that binds to the promoter and creates a “closed” complex
3. Unwinding of DNA forms a transcription bubble, 12-15 bp, to produce an “open” complex
4. Holoenzyme initiates RNA synthesis for ~10 nt

Question 37

Transcription Elongation

1. Dissociation of the __ factor produces the _____ enzyme. “______ ______”
2. ____ replaces ___ subunit
3. Transcription elongation by _____ enzyme ~#-# bases/sec, but slowed by formation of ______ _____
4. Transcription ______: release of ____ and dissociation of _____ enzyme from ____

Answer 37

Transcription Elongation

1. Dissociation of the sigma factor “Promoter Clearage” produces the core enzyme.
2. NusA replaces sigma subunit
3. Transcription elongation by core enzyme ~50-90 bases/sec, but slowed by formation of secondary structure
4. Transcription termination: release of RNA and dissociation of core enzyme from DNA