BIS2A M2 Lecture 17

Central dogma, transcription -- Understand the difference between DNA mutations and DNA damage • Describe the central dogma of molecular biology • Explain why transcription is beneficial • Describe the general process of transcription • Describe similarities and differences between DNA transcription and replication • Describe similarities and differences of transcription in prokaryotes and eukaryotes in terms of RNAP and transcription factors • Understand the differences in prokaryotic vs eukaryotic transcript features and processing

What is the central dogma?

DNA —-(transcription)—-RNA

RNA —-(translation)—-Protein

What are some differences b/w DNA and RNA in terms of the ribose, number of strands, and type of information?

DNA - deoxyirobe (H), double stranded, genetic info

RNA - ribose (OH), single-stranded, transcribed info

Why is RNA less favourable as a genetic information keeper than DNA?

Not stable because OH is more chemically reactive and more susceptible to degrading enzymes

Uracil is the _________ version of Thymine - lost a methyl group

demethylated (not a big problem because the methyl group is not involved in base pair H-bonding)

Describe the process of transcription

First unwind DNA, exposing its bases

Second, use one of the strands as a template to polmerize RNA by base pairing

Third, remove the RNA chain and close DNA

What is the role of RNA polymerase? Which direction does it polymerize?

uses DNA template to polymerize RNA by base pairing from 5’ to 3’

RNA polymerase uses the sequence of the (coding/template) strand to produce a sequence equivalent to the (coding/template) strand.

template — coding

What are the 3 types of RNA?

Messenger RNA - mRNA

Transfer RNA - tRNA

Ribosomal RNA - rRNA

Which RNA’s are involved in transcription? Translation?

mRNA - protein coding transcript

tRNA and rRNA - critical for translation

Note - other types of regulatory RNAs - miRNA, siRNA, and IncRNA, telomerase, splicing

these are all used for gene expression regulation

How many types of RNA polymerase are there in Prokaryotes? Eukaryotes?

Pro - 1 type

Euk - RNApoly1 - rRNA — RNApoly2 - mRNA —RNApoly3 - tRNAs

How does transcription begin?

genes have promoters (DNA sequences)t hat recruit RNA polymerase — it is recruited to the promoter region upstream of the initiation site

What region is a? What is its charge?

Upstream (-)

What region is b? What is its charge?

initiation site (+1)

What region is c? What is its charge?

Downstream (+)

If the promoter is to the right of the initiation site, which direction is upstream? left or right?

right

If the promoter directs RNA polymerase to read to the LEFT strand of the DNA, which strand will be the template?

The upper strand

What are promoters?

DNA sequences that recruit RNA polymerase

Are promoters symmetric or asymmetric?

Sigma factAsymmetric — this determiens which strand is tremplate or coding and sets the direction of polymerization

What binds to the promoter sequences?

sigma factor amino acids

Why are promoters AT rich?

Because these sequences are easier to separate

Typically for bacteria, where are the promotor sequences (in terms of the number scale of upstream to downstream?

-10 element and -35 element

Typically for eukaryotes, where are the promotor sequences (in terms of the number scale of upstream to downstream? What is this promoter called?

-30 element — TATA box

What proteins bound promoters? (protein:nucleic acid interactions)

transcription factors

Transcription factors interact with and recruit RNA pol (?:? interactions)

protein:protein

What are promoters recognized by in bacteria? Euk?

Bacteria: recognized by sigma factor subunit of RNA pol

Euk: recognized by general trancription factors (TFs) that recruit RNA pol

Do differenet eukaryotic RNA Polys share the same promoters and TFs?

No, they’re different for the most part

TFIIx is what

RNA Pol 2 transcription factors

Describe the process of recruitment of RNA Pol II by general TFs

transcription starts. some enzymes like the TBF subunit recognizes the start sequence and binds to the template strand. continue recruiting additional TFIIs as wel as RNA Pol 2. At some point, RNA Pol 2 can free itself from the transcription factors and can continue to elongate the transcript

What features make transciption initiation more complex in euk than prok?

-need several general TFs versus juust one sigma factor

-need more than just promoters and TFs — ex: enhancer seuqences and activator proteins far upstream from gene + mediator linker

-DNA is wrapped around nucleosomes so you need chromation modifying enzymes to increase access to DNA

Does DNA polymerase have proof reading abilities?

Yes it does - proofreads from 3’ to 5’ but its mechanisms are diff

Note: can slow down when wrong base pair is recgonized, wrong nucleotide frayed away from template and rna pol backtracks, cleaving the wrong nucleotide and tries again

What is transcription termination determined by?

terminator sequences

	DNA pol	RNA pol
Type of nucleotide
Direction of polymerization
Need for primer
Proof-reading ability
Additional proteins/factors needed

Add later

How are bacterial genes frequently organized in? What does this mean?

organized in operons - a unit of DNA that contains a cluster of genes controlled by a single promoter

What does an operon lead to?

a polycistronic mRNA, which leads to mutiple proteins

Why are operons advantageous?

Having a single promoter that signals for the synthesis of multiple genes for a certain process is very effective. all the genes you need for that process get synthesized at once.

What does polycistronic mRNA mean?

It’s a signle mRNA that can transcript multiple proteins

Euk mRNAS undergo multiple processing steps before they are ready for translation. These steps help with what?

Help stabilize, protect, and regulate the mRNA and mark it as ready for translation

For euk, transcription happens where? translation?

transcription happens in the nucleus, translation in the cytoplasm

What is added to the 5’ end of the transcript? What does it help mRNA with? How does it protect the transcript? What rols does it also play in?

A 7-methyl-guanosine cap is added to the 5’ end. It helps distinguish mRNA from other types of RNA and protects the transcript from degradation. it also plays a role in export and translation

What is added to the 3’ end of the mRNA? What roles does it play?

a poly-A tail. it helps with stabolity, export, and regulation of translation

What is the difference between an exon and an intron? Also explain how splicing works.

Exon: coding region - gene sequences that get expressed

Intron: non-coding - intervening sequences

splicing removes the introns and joins the exons back together

What does spliceosome do?

it’s a protein:RNA complex that cuts and re-join the mRNAs by recognizing specific sequences that mark the splicing sites

How does splicing allow for protein variety from a single gene? What are the pros and cons?

mRNA can be splied in alt ways and fiff combinations so this leads to related by diff proteins

Useful for variety and efficiency but mis-splicing can lead to disease