(Dr. Heinemann) (Unit C) Topic 18 Flashcards by Lucky Cat

Central Dogma

DNA to RNA to Protein

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Why is there an RNA step in gene expression?

Not required, present for more points for regulation

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Reasons for an RNA step in gene expression

Provides an amplification which contributes to differential gene expression
RNA can be degraded, expression of a gene can be stopped quickly
Provides additional opportunities to regulate gene expression (especially in eukaryotes)

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Consensus sequence

The most frequent base at each position in a group of functionally related DNA elements

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Promoter

DNA sequence required to initiate transcription of a gene or operon

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Terminator

DNA sequence required to stop transcription

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Operon

Set of bacterial genes trancribed from a single promoter and thus expressed from a common RNA

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In bacteria, what is the transcriptional start site called?

+1 site

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What are two consensus sequences in the promoter of bacteria?

-35 and -10 regions

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How do we know that -35 is important?

Making mutations and seeing if it has any effect on transcription

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Bacterial RNA Polymerase

RNAP
* Enzyme that makes RNA transcripts using DNA as a template and nucleoside triphosphates (NTPs) as substrates

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Descibe:

RNAP core enzyme

Comprised of 4 subunits that never separate

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True or False:

RNAP core enzyme can recognize promoters

False, they can only make RNA

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What is promoter specificity of RNA polymerase determined by?

Sigma subunit

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What is the combined polymerase core and sigma subunit known as?

RNA polymerase holoenzyme

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What sequences does sigma make contact with?

-10 and -35 sequences

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Initiation of Transcription:

Step 1

RNAP holoenzyme binds the promoter
* Forms closed complex

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Initiation of Transcription:

Step 2

RNA polymerase unwinds the DNA strands around the start site
* Forms open complex

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Open vs. Closed complex

Open complex occurs with unwinded DNA

Initiation of Transcription:

Step 3

First NTP is brought to the template base pairing with the base at +1

Are primers required for transcription?

Initiation of Transcription:

Step 4

Using ATP, GTP, CTP, and UTP as substrates
* Chain elongation proceeds in a 5’ - 3’ direction, following base pairing rules

What is released when phosphodiester bonds are formed?

Pyrophosphate

Initiation of Transcription:

Step 5

After addition of 5-10 nucleotides, sigma falls off the holoenzyme

# Initiation of Transcription: Step 6

Transcription bubble moves downstream (5'-3') with the template DNA re-annealing behind

# Initiation of Transcription: Step 7

RNA synthesis proceeds until a terminator is reached and RNA polymerase falls off

# Initiation of Transcription: Step 8

Sigma rebinds RNAP and the cycle is repeated

In total, how many steps are there in initiation and replication in transcription?

8 steps

What are the 2 mechanisms for transcirption termination in prokaryotes?

1. Rho-independent 2. Rho-dependent

Rho-independent termination

Also known as instrinsic termination * Terminator sequences can form loop on it self * Pushes transcription bubble off and ends transcription

Rho-dependent transcription termination

Rho protein is attached to RNA * Transcription slows at terminator * Rho catches up and pushes polymerase off of DNA

What is Rho-independent transcription termination dependent on?

Sequence and Secondary structure

What is Rho-dependent transcription termination dependent on?

ATP

How are different promoters transcribed at different levels?

1. Some genes have better -10 and -35 sequences 2. There is more than one sigma factor (each recognizes different promoter sequences) 3. Gene specific regulatory proteins

What are -10 and -35 sequences known as?

Cis element

What are gene specific regulatory proteins known as?

Trans elements * Dynamic

Negative vs. Positive regulation

Negative * Factors repress transcription Positive * Factors activate transcription

Protein - DNA interactions

One alpha helix fits nicely into the major groove of the DNA * Sequence specific interactions * Interactiosn with the DNA backbone

What is the preferred carbon source for E. coli?

Glucose

What happens in E. coli when glucose and lactose is present?

Cell metabolizes glucose and represses use of lactose

What is the purpose of glucose repression or catabolite repression?

Prevents wasteful duplication of energy-producing enzyme systems by repressing transcription of lac & other operons

How is transcription obscurred in lac operon?

Repressor protein blocks the promoter, preventing RNA polymerase from attaching and initiating transcription

Lac operon * Negative control * Positive control

* Negative: Lac repressor (LacI) * Positive: Catabolite activator protein (CAP)

Binding sites of: * Lac repressor * CAP

* Lac operator: DNA element that binds Lac repressor * CAP binding site: DNA element that binds CAP

How does Lac repressor not bind to DNA?

Allolactose - a metabolite of lactose - binds to lac repressor * The allolactose-Lac repressor complex does not bind DNA

What allows CAP to bind to DNA?

1. cAMP - A metabolite of glucose metabolism 2. Low glucose conditions