Chapter 8: RNA and transcription

How is DNA involved in RNA production?

Either one of a DNAs strand is used as a template to form an mRNA strand

What is the DNA strand that isnt being used as a template called?

It is called a coding strand and is a strand that shares the same sequence as the RNA strand being made with the exception for having thymine instead of uracil

What is the promoter region?

A region that acts as a binding site for RNA polymerase and other transcription factors, allowing for transcription to begin

Where is the promoter region located?

In the upstream of a gene (5’ end)

Why does an RNA polymerase rely on a promoter region to bind to a DNA strand to begin transcription?

Its unable to recognize each individual gene on a DNA strand and requires the promoter to begin transcription

What is sigma 70?

A type of RNA polymerase holoenzyme

What occurs in the initiation/elongation of RNA transcription?

A sigma 70 helps direct RNA polymerase to the promoter region the upstream of the gene. Once the polymerase reach the region, sigma 70 will begin the initial unwinding of the template strand and form a transcript bubble, it will then leave the region and RNA polymerase will begin synthesizing an mRNA strand in the 5’ to 3’ direction. Creating an mRNA strand

What is the rate that RNA polymerases synthesizes DNA at?

At a rate of 50-90 nucleotides per second

What are the 2 ways that termination of RNA transcription can occur?

• Through a factor-independent termination called a hairpin structure

• Through a factor-dependent termination called Rho protein

What is the hairpin structure termination?

A structure that forms naturally when hydrogen bonds between 2 sequences in the mRNA strand bind together, triggering the RNA polymerase to dissociate from DNA and terminate transcription

What is the rho protein termination?

Proteins that bind to the RUT site on the mRNA strand and forms a loop, physically disrupting the RNA polymerase and causing it to dissociate from DNA and terminate transcript

What causes degradation of RNA strands?

When a Rnase E cleaves the strand off and the triphosphate group is removed, causing exonuclease to begin breaking down the strand.

Why is the triphosphate group important in RNA strands?

It acts as the protective cap for the strand at the 5’ end, preventing exonuclease from degrading the strand

What is required in addition to RNA polymerase II to begin transcription in eukaryotes?

General transcription factors (GTF) as RNA polymerase cannot bind to the DNA template alone

How do GTFs help RNA polymerase bind to the DNA template in eukaryotes?

By forming a Pre-initiation complex (PIC) that helps recruit the RNA polymerase II

How do GTFs come together to form the PIC?

Carboxy-terminal domain (CTD), the tail end of an RNA poly. II, contains a repeating sequence that acts as a signal to recruit GTFs in order for the PIC to be formed and allow RNA poly. II to begin transcription

How do CID send a signal to recruit GTFs?

A part of the sequence, for example serine, is modified through phosphorylation, allowing for CID to send a signal and be able to recruit GTFs to form PIC

What terminates RNA transcription in eukaryotes?

A CP complex triggers the RNA poly. II to detach from the DNA template and ends transcription

What are the 3 processing steps required in order for mRNA transcript to leave the nucleus IN eukaryotes?

• 5’ cap

• Polyadenylation

• Splicing

What occurs in the 5’ cap process?

A GTP cap is placed at the 5’ end of the mRNA transcript in order to prevent decay and be able to associate with ribosomes

What occurs in polyadenylation?

A CPSF complex enzymatically adds 50-250 adenosine to the 3’ end of the mRNA transcript, forming a poly-A tail, which prevents decay and promotes translation by interacting with the translation machinery

What are introns?

The in-between sequences that are non-coding and separate genes

What are exons?

the coding sequences that are being expressed and used as templates

What occurs in Splicing

Introns are removed from the mRNA transcript by snRNAs, leaving only exons

What are the ribozymes that are used to remove introns?

Splicesomes that contain snRNAs that remove the introns

What are the splice sites that introns contain?

They are boundaries of GU and AU sequences that shows where snRNAs have to remove the introns from

What is alternative splicing?

A type of splicing where introns AND some exons are removed, with the remaining exons being joined together to form different pattern and resulting in lots of variations in mRNA sequences, which eventually leads to variations in proteins

What are protein isoforms?

the different variations of proteins that are generated from variation of mRNA sequences

What does the mRNA transcript after processing consist of?

A 5’ cap, a 3’ poly-A tail, and exons only that is able to move into the cytoplasm

How does the decay of an mRNA transcript occur in EUKARYOTES?

An deadenylase removes the 3’ poly-A tail and the 5’ cap, causing the transcript to break down in both 5’-3’ and 3’-5’ direction, becoming a nucleotide that can potentially be reused to form a new RNA

What is the half-life of a eukaryotic RNA?

Starts between 20-30min up to 20-24hrs

What is RNA inference (RNAi)?

A strand that has the ability to target specific RNA strands and silences them as a regulation of gene expression

What was the 1st experiment performed that begun the discovery of RNAi?

Scientists took a gene of purple flower and used transposal, expecting dark petals and resulted in white strips of petals instead. Leading to speculation on what was interfering with gene expression

What was conclusion made from the 1st experiment relating to RNAi?

When genomes are randomly inserted, rather than being expressed, its hidden in the promoter region and leads to there being dsRNA

what was the 2nd experiment performed that helped lead to the discovery of RNAi?

Scientists injected worms with ssRNA and dsRNA, the results showed dsRNA formed a defense for the worm and silenced the unc-22 gene within the worm.\

What was the 3rd experiment performed that lead to the discovery of RNAi?

A viral gene was inserted into a plant and was expected to cause issues, however instead the plant remained healthy as the viral gene caused dsRNA to be produced and was silenced by the dsRNA

What occurs in the RNAI pathway?

dsRNA is introduced to the cell and is cut by dicer enzymes into siRNAs, enabling the assembly of a RISC complex. Once assembled, siRNAs are loaded into the complex and has its passenger strand removed by argo2 proteins, leaving only the guided strand responsible for silencing mRNA

how do siRNAs silence target mRNA?

It guides the RISC complex to the target mRNA and once found, the complex cleaves off the mRNA at a specific point using argo proteins, causing it to degrade

What are siRNA’s?

Double stranded RNA that contains 21-nucleotides with each end having a 2 nucleotide 3’ overhand

• contains a passenger strand and a guided strand

What is the passenger strand of siRNA?

A strand that has an identical sequence as the target mRNA

What is the guided strand of siRNA?

A strand that has complementary sequences to the target mRNA