Transcription - Lecture 7

Transcription (i.e. RNA synthesis)

required for gene expression

makes genes accessible

the central dogma

central information pathway in a cell

DNA makes RNA, RNA makes proteins

Where transcription occurs

occurs on a per-gene (or small group of genes) basis

Transcription - RNA synthesis

this does not require DNA synthesis to happen beforehand

Translation (i.e. protein synthesis)

transfers genetic information to proteins

DNA sequences

serve as physical and functional units of inheritance

DNA sequences - genes

people have two copies of most _____, but this number can vary

DNA sequences - variation

major source of _________ between individual genomes

DNA in somatic cells

in multicellular organisms, DNA is identical in all of these cells, but gene expression differs within the +200 different cell types

Gene expression

connects genotype (DNA) to phenotype (appearance)

Disease

often connected to changes in default gene expression level

Synthesis of RNA (transcription) - direction

unidirectional as only one DNA strand used as a template

Synthesis of RNA (transcription) - location

occurs in cytoplasm (prokaryotes) or nucleus (eukaryotes)

Synthesis of RNA (transcription) - transcription start sites

this is at a specific DNA location

Synthesis of RNA (transcription) - "transcription unit"

each of these encodes at least 1 gene

Transcription stages

initiation, elongation, and termination

Denaturation

transcription requires DNA to undergo this

strands must be separated for cellular machinery to access nucleic acid

Denaturation location

occurs at a local region; entire helix does not unwind

B form to A form

DNA makes this transition as this conformation improves fidelity via template nascent and protein-DNA interactions

prokaryotes - RNA polymerase

in prokaryotes ___ __________ has helicase activity

transcription factor IIH

in eukaryotes, this functions as a helicase for RNA polymerase II

RNA polymerase

key DNA-binding protein that catalyzes phosphodiester bridge formation in RNA

RNA polymerase - ribonucleotides

added to the 3' end of newly synthesized

Prokaryotic RNA polymers

E. coli has a single polymerase

synthesizes all transcribed products (rRNA, mRNA, tRNA)

Eukaryotic RNA polymerase

humans have three (I, II and III)

different polymmyerases synthesize different RNA products (e.g., Pol II generates mRNA)

Important aspects of RNA synthesis

1. requires all four activated ribonucleotides and Mg2+ or Mn2+

2. New RNA strand has complementary sequence to template DNA strand

3. RNA polymerase does not require a primer

4. Errors generally not corrected by RNA polymerase; little proofreading

Prokaryotic RNA synthesis: Initiation - promoters

special DNA sequence located upstream of the start site

Prokaryotic RNA synthesis: Initiation

promoters direct RNA polymerase to transcription site

may contain 1 or more upstream promoter (UP) element

Prokaryotic RNA synthesis: Initiation site

in prokaryotes, known as -10 and -35

Prokaryotic RNA synthesis: Initiation - transcription factors

A DNA-binding protein; may also bind ligands (signal-sensing)

play an important regulatory role

ligands

molecule that binds another (generally larger) molecule

Prokaryotic RNA synthesis: Initiation - activator

if bound to a promoters upstream region, then activate transcription by recruiting RNA polymerase

Prokaryotic RNA synthesis: Initiation - repressor

if bound to a promoter, then suppress transcription by interfering with RNA polymerases access to start site

Prokaryotic RNA synthesis: Initiation - E. coli RNA polymerase

has 5 subunits (α2ββ'ωσ; holoenzyme)

Prokaryotic RNA synthesis: Initiation - sigma subunit

locating the promoter depends on this

- decreases enzymes affinity for general DNA regions

- allows RNA polymerase to "scan" for promoter along DNA

Prokaryotic RNA synthesis: Initiation - core enzyme

once located, sigma dissociates and ____ ______ (α2ββ'ω) remains

Prokaryotic RNA synthesis: Elongation

Core enzyme elongates RNA strand in a 5' to 3' direction

transcription bubble has polymerase, DNA template and nascent RNA

Prokaryotic RNA synthesis: Elongation - Nascent RNA

newly formed primary transcripts RNA

this forms a hybrid helix with ~8 base pairs of template DNA

Prokaryotic RNA synthesis - Termination

transcription terminated by synthesized RNA product of stop signals

not due to stop signals themselves within the DNA template

Prokaryotic RNA synthesis: Termination - Rho factor (p)

about 20% of prokaryotic termination events involve this factor

Rho factor (p)

an RNA binding protein that causes RNA polymerase to dissociate

Prokaryotic RNA Synthesis: RNA polymerase generated primary transcripts

Messenger RNA (mRNA)

Transfer RNA (tRNA)

Ribosomal RNA (rRNA)

Final products of prokaryotic RNA synthesis

RNA polymerases can generate three different primary transcripts

Nucleases

components of RNA synthesis (mRNA, tRNA, rRNA) excises by _________; may undergo further processing

How does eukaryotic RNA synthesis differ? - mechanistically

mechanistically similar in prokaryotic and eukaryotic organisms

How does eukaryotic RNA synthesis differ? genome size

genome size necessitates multiple promoters, with human genome estimated to have +9,000 across all chromosomes

Key differences in eukaryotic RNA synthesis

abundance of enhancer sequences

extent to which RNA product further processed / edited

abundance of "junk DNA"

Abundance of enhancer sequences - regulatory DNA sequences

these sequences (~50 - 1500 bp long) that are transcription factor binding sites

transcription factor binding sites

cause DNA to bend, bringing enhancer closer to promoter

help recruit RNA polymerase to the gene

Abundance of enhancer sequences

stimulate transcription over long distance (10^6 bp)

transcription over a long distance

may be upstream or downstream of gene

effective within introns or on either DNA strand

But must be within same chromosome as gene

enhancer sequences in prokaryotes

also in prokaryotes but much less abundant

intron

non-protein coding regions

RNA product further processed / edited

following intron removal, eukaryotic transcripts selectively methylated for stability and recognition

RNA product further processed / edited - mRNA transcripts

these also capped (5' end) and have a poly A tail added (3' end) to promote translation

alternative splicing

this is used to increase proteome complexity

proteome

complete set of proteins expressed in a cell

Abundance of "junk DNA"

majority of eukaryotic DNA is non-protein coding

Why we have junk DNA

regulatory sequences (like promoters and enhancers)

protection from exonuclease (e.g., telomeres)

DNA that generates other transcribed products (tRNA and rRNA)

non-coding prokaryotic DNA

minority (<14%) of this DNA is non protein coding

mitochondrial genome

has 37 genes

mitochondrial 37 genes

13 for protein components of the electron transport chain and ATP synthase

22 for tRNA

2 for rRNA

mitochondrial transcription

resembles prokaryotic process, as a single polymerase synthesizes all transcribed products but two

Two exceptions to what a single polymerase synthesizes in the mitochondria

- polymerase (POLRMT) is not multi-subunit (i.e., no sigma subunit)

- termination involved mTERF protein, which induces base flipping in DNA

true

true or false

RNA synthesis does not require DNA synthesis to happen beforehand

false

true or false

DNA is identical in all somatic cells, and gene expression is the same within the +200 different cell types

false

true or false

RNA polymerase has strong proofreading capabilities like DNA polymerase

E.) all of the above

RNA polymerase can generate a primary transcript containing

A.) rRNA

B.) tRNA

C.) mRNA

D.) none of the above

E.) all of the above