Prokaryotic Transcriptrion FINAL

A gene is not just the protein-coding sequence- what two major parts does it include?

• coding region

• regulatory elements

what is the coding region in a gene

the actual DNA sequence that will determine the amino acid sequence ( primary sequence) of a protein

what are some examples of regulatory elements within a gene

• promoter

• enhancers/silencers

• transcription factor binding sites

A gene is used to create an RNA primary transcript. What is the mechanism for this?

1. RNA polymerase binds the promoter

2. DNA strands separate

3. One strand (template strand) is used to build RNA

4. RNA is synthesized 5’ → 3’

Yielding a primary transcript

what is the primary transcript

the inital RNA copy

When a primary transcript is made for bacteria, the RNA that is first made is already usable by ribosomes, therefore there is no editing phase, unlike in eukaryotes that have pre-mRNA that needs to be edited. Why?

because they dont have a nucleus- so transcription and translation happen in the same space ( the cytoplasm) - so as soon as RNA is made ribosomes jump on it therefore making the primary transcript the functional mRNA

In eukaryotes primary transcript is NOT immediately translation ready - why is this?

because transcription happens in the nucleus, but translation happens in the cytoplasm

In eukaryotes primary transcript is NOT translation ready because transcription happens in the nucleus, but translation happens in the cytoplasm.

Therefiore the mRNA has to pass through the nuclear membrane via nuclear pores

What is the critical issue with this?

The nucleus does NOT allow incomplete or faulty RNA to leave. Therefore the processing steps are like a checklist to earn permission to exit the nucleus

what is a continous sequence within the coding region of a gene from start codon to stop codon that is a potential protein coding stretch that has a start codon , ends at a stop codon , and has no stop codons in between

open reading frame

what is a cistron

a gene that encodes for one protein

where are cistrons found?

in open reading frames which are found inside of the coding region of the gene

In bacteria, one mRNA can contain multiple cistrons. What does this mean?

one primary transcript yields multiple proteins

In bacteria, one mRNA can contain multiple cistrons therefore making it polycistronic. Why?

because genes involved in the same pathway are grouped together in operons

In eukaryotes one mRNA encodes for only one protein, what does that make it’s mRNA

monocistonic

Mainly in eukaryotes one mRNA yields one protein-making it monocistronic. Why is this and what does it allow?

because each gene has its own promoter and is transcribed independently therefore allowing much more precise regulation

True or False coding strand sequence is the actual blueprint for amino acids to make proteins

true

“ALL CDS (coding sequences) are ORFs but not all ORFs are CDS”

How are all CDSs ORFs

they have:

• start codon

• stop codon

• and are continuous, fitting the definition of an ORF

“ALL CDS are ORFs but not all ORFs are CDS”

How are all ORFs not CDSs

the genome has many random ORFs but most are not actually used becasue they have no promoter, not transcribed, not translated, or have regulatory signlas missing

therefore ORFs have potential BUT CDS are confirmed, functional protein coding regions

What the big picture connection for genes?

• A gene contains coding + regulatory info

• It is transcribed → primary RNA

• In:

  • Bacteria → ready immediately

  • Eukaryotes → must be processed

• The RNA contains:

  • ORFs (possible proteins)

  • CDS (actual protein-coding regions)

• Depending on organism:

  • Prokaryotes → multiple proteins per mRNA (polycistronic)

  • Eukaryotes → one protein per mRNA (monocistronic)

what is a group of genes controlled together with the key idea of multiple genes being on one contol system found in prokaryotes?

operons

why are operons useful

If a cell needs a pathway (like making ATP), it needs all the proteins together, not one at a time therefore ensuring efficieny coordination

which direction are operons arranged in

5’ → 3’ all lined up one after another - being transcribed in one continuous pass

what would be the sequence of ORFs

Promoter → ORF1 → ORF2 → ORF3 → Terminator

In an open reading frame, one promoter and one terminator yields a polycistronic RNA how does this happen?

Rna polymerase binds one promoter and transcribes straight through multiple ORFs, stopping at one terminator

even though it ones prokaryotic mRNA, it doesnt make one protein , what does it make

multiple indpendent translation units

How is an atp operon in e.coli an example?

the atp operon contains 9 ORFS and encodes subunits of ATP synthase- they are all grouped becasued they all work together at the same time

Why do cis and trans-acting factors matter for transcription?

transcription only happens when cis elements are present- meaning there must be a promoter/operator DNA sequence and when trans factors bind those DNA sequences to activate or repress transcription

Transcription does not always require both cis elements and trans factors to start but rather that…

cis elements define where transcription can happen and trans factors decide whether it actually happens

what are transacting factors usually

DNA- binding proteins

transacting factors are typically DNA binding proteins which are usually known as

transcription factors

the process of DNA template-dependent RNA synthesis and is catalyzed by RNA polymerase

Transcription

While DNA replication replicates the entire chromosome/genome… what is the difference with RNA synthesis

RNA synthesis much more selective

When does transcription start and end

it starts at a promoter and ends at a terminator

what is the finished RNA molecule after transcription

the primary transcript

what is the transcription start site defined as

the exact first nucleotide where RNA synthesis begins

where is the transcription start site always located

+1

what is important to know about +1

it is NOT the promoter, it is the first base that gets copied into RNA

how is the start of transcription always at +1 if transcription starts at the promoter?

The promoter recruits RNA polymerase, but the +1 site is where RNA synthesis begins.

RNA polymerase binds the promoter, but transcription starts at +1.

There are directional terms based on transcription movement.

What is upstream?

• where polymerase came from

• regulatory control region

• usually noted by (-)

There are directional terms based on transcription movement.

What is downstream?

• where polymerase is going

• coding region of a gene

what does it mean when something is proximal?

• close to the gene

• often near promoter

what does it mean when something is distal?

• Far away from gene

• Can still regulate gene (especially in eukaryotes via enhancers)

What is something RNA polymerase can do that DNA polymerase cannot. DNA polymerase needs this for replication and RNA polymerase does not

Rna polymerase can start synthesizing RNA de novo— without a primer

why does it matter that RNA polymerase can start synthesizing de novo

because it can start the first bond on its own and initiate RNA synthesis directly at +1 which is why transcription can begin immediately at a gene

what are the substrates of RNA polymerase

NTPs (ribonucleoside triphosphates) such as ATP, GTP, CTP, UTP

the substrates of RNA polymerase are NTPs what does each one contain

• a sugar

• a base

• three phosphate groups

RNA polymerase links nucleotides together by forming what kind of bonds

phosphodiester bonds

How do the phosphodiester bonds form?

the 3’ OH of the growing RNA chain attacks the incoming NTP and two phosphates are released (pyrophosphate)

what type of base pairing does RNA polymerase use

watson-crick base pairing

what is the big difference between DNA polymerase and RNA polymerase

there is no proofreading in RNA polymerase

Since theres no proofreading in RNA polymerase what is the error ratew

much higher

what is the main flow of transcription

RNA polymerase binds promoter

↓

positions at +1

↓

uses DNA template strand

↓

selects NTPs via base pairing

↓

forms phosphodiester bonds

↓

RNA elongates 5’ → 3’

↓

no proofreading → occasional errors

What does the RNA transcript resemble

the coding strand

How does RNA temporraily base pair with DNA

during transcription RNA polymerase opens a small region of DNA and the new RNA being made briefly pairs with the DNA template

During transcription:

• RNA polymerase opens a small region of DNA

• The new RNA being made briefly pairs with the DNA template

What does this create?

a temporary RNA-DNA hybrid helix until RNA peels away and the DNA re-closes

what is the template strand?

the dna strand RNA polymerase reads

what does RNA polymerase use the template strand for?

to decide which NTP to add

what is the noncoding strand?

it is not used for copying, but matches the RNA sequence

the nontemplate (noncoding) strand is NOT used for copying, but matches the RNA sequence - why does it match RNA?

because RNA is complementary to the template strand and the coding strand is already complementary to the template- the only difference is that RNA uses U instead of T

what does it mean when the coding strand can be in either DNA strand?

DNA is double stranded so for one gene the top strand might be coding and for another gene the bottom strand might be coding - therefore there is NO universal coding strand

what does rifampin target

it inhibits bacterial transcription by binding RNA polymerase

what does rifampin specifically bind to in RNA Polymerase

the β subunit which is essential for RNA synthesis

RNA polymerase binds the promoter, starts transcription at +1 and builds RNA from NTPs. This is essential for making mRNA, making proteins, and keeping the cell alive.

What does rifampin do to this process?

rifampin binds RNA polymerase before or during early transcription and blocks initiation- this prevents RNA polymerase from properly extending the RNA chain after starting

what is the result of rifampin

RNA cant be made and proteins cannot be made and bacteria die

what is the key idea behind rifampin

no transcription means no gene expression which means no survival of bacteria

how does rifampin work clinically

the bacteria relies heavily on transcription to survive in host cells and respond to stress—if you were to block (rifampin) transcription it would create a powerful antibiotic effect

How does resistance to rifampin happen

when theres a mutation in the rpoB gene

what is rpoB

a gene that encodes the β subunit of RNA polymerase

what happens when β subunit of RNA polymerase mutates?

• The structure of the β subunit changes slightly and rifampin can no longer bind effectively allowing RNA polymerase to still work normally for bacteria

How does the use of Rifampin tie everything together?

RNA polymerase builds DNA from DNA so it need to initiate transcription at promoters. Rifampin blocks initiation at promoters. This shuts down RNA synthesis

Why doesnt rifamplin kill human cells

there are structural differences with bacterial rna polymerase and eukaryotic rna polymerase. Rifamplin fits bacterial enzymes and does NOT effectively bind human enzymes

Bacterial RnA has 2 modes- what are they

• core enzyme

• holoenzyme

what does the core enzyme do?

build RNA (elongation)

what does the holoenzyme do?

finds where to start (initiation)

what makes up the holoenzyme

the core and the σ

Unlike eukaryotes (which have multiple RNA polymerases) for transcription, what does bacteria like e.coli use for transcription?

one enzyme for all transcription

Unlike eukaryotes (which have multiple RNA polymerases), bacteria like E. coli use one enzyme for all transcription. So what does it have to do?

find promoters, start transciption, and elongate RNA- thats why it uses different subunits to specialize tasks

the core enzyme is the RNA building machine- what makes up the core enzyme

α₂ β β′ ω

what does the core enzme do specifically?

Elongation

what does elongation do?

add nucleotides to RNA

what do the 2 copies of α do as the core enzyme subunit?

they help assemble the enzyme and interact with activator proteins for regulation

what does the β and β′ (beta and beta prime) do as the subunit in the core enzyme?

form the catalytic center and actaully link NTPs together- forming the phosphodiester bonds and growing the RNA chain

what does ω do in the RNA subunit?

stablizes the structure

what is the key idea for the core enzyme

it can MAKE RNA, but it doesnt know where to start

what does the holoenzyme do?

it starts the transcription correctly

what does the σ do in the holoenzyme

σ recognizes promoters

Why is it so important that σ recognizes promoters

it tells RNA polymerase where a gene begins and where to bind DNA

What is the mechanism of the holoenzyme

• σ binds to specific DNA sequences (promoter)

• RNA polymerase is positioned correctly

• Transcription begins at +1

What happens if we dont have a σ

RNA polymerase would bind DNA randomly with no proper gene expression

what happens after initiation

the first 10 nucleotides are made with the sigma often falling off and the core enzyme continuing elongation

What is the flow if initiation?

Holoenzyme (with σ) → finds promoter → starts RNA

↓

σ leaves

↓

Core enzyme → continues elongation

different σ factors mean different genes which means not all promoters are the same. So what does this mean for bacteria?

Bacteria have multiple σ subunits and each recognizes different promoter sequences

Different σ factors mean different genes which means not all promoters are the same. This means that bacteria has multiple σ subunits and each recognizes different promiter sequences. What is a primary example of this

σ⁷⁰

what is significant about this σ⁷⁰

it is a default promoter which is a housekeeping gene

what do the other σ factor do?

they respond to stress and heat shock

why is it important to have different σ

it allows bacteria to rapidly switch gene expression and respond to the environment. By changing σ, you change which genes are transcribed

due to promoters being within the DNAsequence what can you classify promoters as

cis elements- DNA sequence

since having a σ factor determines if transcription properly occurs what can you classify σ factors as

trans factor - proteins that recognize it

The core RNA polymerase ____________, while the σ subunit_______________________________

synthesizes RNA; allows the holoenzyme to recognize promoters and initiate transcription at the correct site

what is a prokaryotic promoter

a DNA region (on the coding strand) that tells RNA polymerase where to bind and start transcription.