MBIO 2020 / Topic 3: Transcription

What is transcription?

Synthesis of RNA using DNA as a template.

What does the primary structure of RNA look like? What does the secondary structure of RNA look like?

• Primary: RNA predominantly exists in single strands.

• Secondary: RNA folds back upon itself in regions where complementary base-pairing is possible.

Differentiate the three major types of RNA in regards to their structure and how it affects how long they could live.

• mRNA are typically only in primary structure so exists only for a few minutes before being degraded by ribonucleases.

• tRNA and rRNAs long-lived because secondary structures (the folding on itself) shields them against ribonuclease attack.

What does RNA pol catalyze? Where do they get this energy?

RNA pol catalyzes phosphodiester bonds between the ribonucleotides ATP, GTP, UTP, and CTP, using the energy released from the hydrolysis of two energy-rich phosphate bonds of incoming ribonucleoside triphosphates.

How does the chain of RNA elongate? What is the direction of RNA synthesis?

Ribonucleoside triphosphates are added to the 3’ hydroxy of the ribose of the preceding nucleotide, thus RNA synthesis is 5’ to 3’ (and antiparallel to the DNA template strand it was transcribed from).

Differentiate the phrases template strand, non-template strand, non-coding strand, and coding strand in a way that makes sense in respect to RNA pol activity.

• RNA pol reads only one strand of DNA (the template strand). The other strand is not read (the non-template strand).

• RNA pol reads one strand that does not have the code for the protein to be made (the non-coding strand). The other strand has the code for the protein (the coding strand).

Why is the template/non-coding strand read instead of the non-template/coding strand?

What we want is to copy the gene. If we were to copy the coding strand, then we would get a copy of base sequences that the gene would base-pair to and not a copy containing the bases of the gene. We need to get a copy containing the bases of the gene. The only way to do that is to copy the non-coding strand. If we read the non-coding strand, then we would be able to get the bases of the coding strand, which is what we want.

what are the two pieces of the transcription apparatus?

RNA pol + σf

• What is the RNA pol also called in airport-speak? What does it do?

• What is the sigma factor also called in airport-speak? What does it do?

• Also called the plane, it is the large multimeric core enzyme responsible for catalyzing RNA synthesis.

• Also called the pilot, it is the protein controlling the binding of RNA pol to promoter region.

where is the promoter region on DNA? what does it contain?

> located upstream of a gene

> contains -35 and -10 boxes

when does the σf detach?

detaches once the first few RNA nts linked

why are -35 and -10 boxes conserved?

> more conserved

> more “standardized” [recognizable binding site for RNA pol]

> more efficient binding

What is more conserved, the -10 or -35 box? What explains this difference in conservation?

• -35 box: While it's still conserved for efficient RNA pol binding, it can show slight variations to accommodate different sigma factor.

• -10 box: Highly conserved for DNA melting. The -10 box is AT-rich to form the transcription bubble easily. If all transcription requires DNA melting and bubble formation, and if it requires an AT-rich region, then the -10 box must be highly conserved with AT-richness. There’s no other reason (in general) for the -10 box to vary differently from being AT-rich and dissimilar from being an AT-rich sequence.

• σ⁷⁰ RpoD

• σ⁵⁴ RpoN

• σ³⁸ RpoS

• σ³² RpoH

• σ²⁸ FliA

• σ²⁴ RpoE

• σ¹⁹ FecI

• σ⁷⁰ RpoD: Major housekeeping sigma factor for normal growth.

• σ⁵⁴ RpoN: Nitrogen assimilation.

• σ³⁸ RpoS: Stationary phase, plus oxidative and osmotic stress.

• σ³² RpoH: Heat shock response.

• σ²⁸ FliA: For genes involved in flagella synthesis.

• σ²⁴ RpoE: Response to misfolded proteins in periplasm.

• σ¹⁹ FecI: For certain genes in iron transport.

During transcription, what are the five distinct regions on DNA? Differentiate them.

• Promoter: Recognition and binding region for RNA pol holoenzyme.

• Leader: Region transcribed into mRNA but not translated.

• Coding: Region transcribed and translated into polypeptide, wherein transcription begins at position +1, which is usually an A or G.

• Trailer: Region transcribed into mRNA but not translated and prepares RNA pol to release the template/non-coding strand.

• Terminator: Region where RNA pol detaches from the template/non-coding strand.

explain how the RNA pol recognizes and binds to promoter

> σf (not core enzyme) recognizes + binds to -35 on coding strand in 5’ to 3’

> σf (not core enzyme) recognizes + binds to -10 on coding strand in 5’ to 3’

> RNA pol binds to non-coding strand (3’ to 5’)

+ back wheels of plane land first (to -35) and then the front wheels (to -10)

what determines promoter strength?

how conserved the sequences in -35 and -10 are

• What is the closed complex in transcription initiation? When does this form?

• What is the open complex in transcription initiation? When does this form?

• The closed complex is where DNA is still double stranded. This forms when the RNA pol recognizes and binds to promoter on the onset of transcription initiation.

• The open complex is where DNA is now unwound into ssDNA. This occurs when the -10 is melted, a transcription bubble is formed, and mRNA chain starts to be polymerized, starting at the +1.

• How many bp is usually unwound within the open complex?

• During RNA transcription, where and when do the open complexes form?

• During RNA transcription, where and when do the closed complexes form?

• What is the dsRNA/DNA hybrid referring to?

• Around 16-20 bp is unwound within the open complex.

• The open complex is formed where RNA pol is, where addition of rNTPs are and where the elongation of the chain of rNMPs is occurring.

• The closed complex forms behind RNA pol during its activity, as the DNA re-anneals after its segment of DNA has been finished transcribing from the transcription bubble.

• The dsRNA/DNA hybrid refers to a hybrid molecule formed during transcription, where one strand of DNA and one strand of RNA are paired together. Note: The hybdrid does not include the RNA transcript that has separated from the template strand.

Is it only that one round of transcription can occur at a time? Why or why not?

When conditions permit, transcription is a repetitive process. Initiation is not dependent on completion of previous round of transcription.

How does a hairpin structure form? What does this structure lead to in the grand scheme of transcription?

• Inverted repeats at the end of gene sequence in the coding strand flank a non-repeating unit.

• When the mRNA is transcribed, the RNA will flap about and will form a hairpin/stemloop structure.

• This structure leads to transcription termination.

What are the two mechanisms by which RNA pol can be removed from the template strand?

Rho-dependent (enzyme-dependent) and Rho-independent (self-termination).

• Is the hairpin structure present in Rho-dependent or Rho-independent mechanisms? Trick question.

• Are inverted repeats present in Rho-dependent or Rho-independent mechanisms? Trick question.

• How do hairpins lead to termination?

The hairpin structure is present in both Rho-dependent and Rho-independent mechanisms. On that note, invented repeats can happen in both. These structures present a physical obstruction to cause RNA pol to temporarily stall.

explain how the Rho-independent mechanism of termination works

> hairpin slows transcription of RNA pol

> A-rich region present after inverted repeats in non-coding

> RNA pol synthesizes mRNA region with multiple succeeding U’s

> AU bonds in dsRNA/DNA hybrid and weak compared to GC

> relatively destabilizes hybrid

> RNA pol falls off the template w/ mRNA.

what does the Rho-dependent mechanism require?

> rho factor protein + rut site on mRNA

• Does the rut site code for the rho factor? What’s the relationship between the rut and rho?

• Is the rut site active in RNA or DNA? Feel free to clarify.

• The rut site doesn’t make the rho factor, but it gets bound and recognized by the rho factor.

• The rut site is active only in RNA, but encoded in DNA.

explain how the Rho-dependent mechanism of termination works

> rut site transcribed

> multi-Rho hexameric protein assembles around and binds to rut

> rho moves 5’ to 3’ via helicase activity via ATP hydrolysis

> hairpin slows transcription of RNA pol

> rho catches up to RNA pol, pushes it off non-coding, and destabilizes hybrid

> hybrid releases RNA pol and mRNA