Topic 8 - BSCI222 EXAM 2

Central Dogma

info flows in 1 direction: DNA → protein; (DNA → RNA → mRNA → proteins)

Composition of DNA vs RNA: Chemical:

RNA: ribose has OH (blue) at 2’ carbon, DNA: deoxyribose doesn’t; DNA: Thymine, RNA: uracil replaces Thymine

Composition of DNA vs RNA: Physical:

RNA: single stranded, DNA: double stranded; RNA: form non-helix secondary structures (hairpins) DNA: doesn’t

RNA is an unstable intermediary:

temporary product that carries this genetic info to make proteins

Transcription:

making single stranded RNA from double stranded DNA

Transcription prokaryotes vs eukaryotes:

Prokaryotes: not much processing; Eukaryotes: needs processing before transcription takes place

Transcription: which strand is the template?

depends on gene being transcribed

Directionality in transcription:

very important (DNA Antiparallel: 1 runs in 5’ to 3’ & 1 in 3’ to 5’) RNA polymerase reads DNA template strand in

RNA transcript is built in

5’ to 3’ direction

Nontemplate strands also known as:

sense/coding strand

Template strand also known as:

antisence/non-coding strand

transcription unit:

stretch of DNA involved in making RNA

Transcription start site ALWAYS at

+1 position

Nucleotide before +1 is

-1 position

Is there a 0 position?

No

Upstream:

left is upstream (-) towards promoter

Downstream:

right is downstream (+) away from promoter (direction RNA pol will be moving)

Terminator:

DNA sequence involved in ending transcription - last position where RNA transcription takes place

Transcription - Initiation

1. Promoter recognition: recognize gene that needs transcription (bc transcription doesn’t copy the whole genome!)

2. Forms transcription bubble (DNA opens & forms bubble (expose template strand for RNA pol))

3. Creation of first bonds of RNA: RNA nucleotides join to make a new RNA transcript (denovo bc 3’OH not needed like DNA synthesis)

Consensus sequences: 2 important regions

-35: 5' TTGACA 3’ (initial recognition & binding to promoter); -10: 5' TATAAT 3’ (allows for DNA to unwind & transcription bubble to form)

Sigma factor is bound to

RNA polymerase (essential to identifying promoter)

Bacterial Promoter: -10: Variation affects

binding efficiency & gene expression!

Transcription - Prokaryotes

Holoenzyme:

Core enzyme + sigma

Sigma:

promoter recognition → binds -10 & -35 region to initiate transcription

When transcription starts the Core Enzyme (RNA polymerase), what happens with sigma?

sigma ensures that core enzyme binds in a stable manner, sigma releases & dissociates from core enzyme → without sigma, has an affinity for DNA but cannot distinguish between promoter sequence and other DNA sequences

Initiation

Holoenzyme initiates formation of transcription bubble (12-14 nt)

RNA synthesis begins with triphosphate nucleosides (energy!!!)

RNA polymerase opens up DNA (double stranded) in -10 region → makes transcription bubble, RNA synthesis begins → starts to join nucleotides

Elongation

sigma released (moves beyond promoter & core enzyme does RNA synthesis)

RNA pol moves through DNA(maintains transcription bubble ~12-14 nucleotides); within bubble: DNA-RNA hybrid ~8-9 bp

RNA separates from DNA(DNA strands join back together)

Termination

Rho-dependent/extrinsic terminators & Rho-independent/intrinsic terminators (Both use hairpin loops in RNA to pause RNA pol → Physical blockage!)

Rho-dependent terminators

Depends on Rho protein: helps release RNA transcript; (requires additional protein for termination)

Rho-independent terminators

RNA sequence allows for termination (does not require an additional protein)

Rho - dependent termination

signal sequence (rut): rho binds on transcript, Rho (uses ATP to get closer to RNA pol)

secondary structure (hairpin & loop) “slows down” RNA pol so Rho can catch up

Rho has helicase activity (RNA pol hits terminator/hairpin → disrupts DNA/RNA hybrid = releases RNA transcript)

Rho - independent termination

secondary structure causes RNA pol to “slow down”; an adjacent A/U-rich region at end (U-A or A-T hybrid) weak so it detaches bc of weak connection & transcription ends)

core promoter:

where RNA pol binds & starts transcription

Promoters can be in which positions?

negative or positive

Transcription - Eukaryotes

Eukaryotic Transcription

3 or 4 RNA polymerases (I, II, & III, some have IV); each specializes on different type of RNA; each has a different combination of promoter consensus sequences that they are able to recognize

To transcribe a gene, Eukaryotic RNA Pol II need help from:

“Transcription Factors” & Promoter

“general transcription factors” bind

Core Promoter

“general transcription factors”

help RNA pol II assemble at right location & start transcription; Binds Basal transcription factors: proteins & RNA pol II = enough to initiate minimal (“basal”) levels of transcription

“specific transcription factors” bind to

regulatory DNA sequences (Regulatory Promoter)

“specific transcription factors”

can increase or decrease transcription; Binds Regulatory transcription factors: bind to specific sequences to bring about a higher level of transcription by stimulating basal apparatus.

TFIID (TBP)

recognizes & binds to TATAA box (in core promoter region) → marks when transcription will begin

TFIIA & TFIIB

bind & stabilize complex so machinery can easily bind & land correctly

TFIIF + RNA pol II

bind & helps recruit & stabilize polymerase at promoter

TFIIE recruits

TFIIH

TFIIE

binds to transcription initiation complex, opens jaws of RNA pol

TFIIH

binds & uses ATP to unwind DNA at transcription start site → acts as helicase (allows DNA template to open up so RNA pol can access template strand)

RT-qPCR

estimate how much RNA from a specific gene is in a sample

RT-qPCR: FOR ONE GENE!

Collect RNA samples from tissue of interest (quantify how much total RNA you are starting with)

reverse transcriptase: RNA into cDNA, put sample into a machine with sequence-specific primers (uses fluorescence to see how much cDNA is present)

Measures fluorescence every time particular cDNA is accumulating in PCR cycle to determine how much RNA of that gene relative to a standard.

mRNA sequencing on illumina/sequencing “transcriptome” FOR ALL GENES:

1. collect RNA sample from tissue of interest

2. reverse transcriptase to make cDNA

3. do illumina sequencing

4. use computational biology (bioinformatics) to identify how much of each mRNA transcript is present in sample

5. end with millions of short sequences from sample

NOT ALL genes encode a protein, Non-coding RNA (ncRNA) is….

Non-coding RNA (ncRNA) is transcribed, but not translated (don’t code for proteins)

important functions: rRNA, Telomerase, snRNPs (splicing), piRNAs (silencing); Some are functional RNAs that interact with mRNAs: tRNA, miRNA, siRNA, lncRNA, & sRNA

In eukaryotes where does transcription occur? What has to happen before translation?

in nucleus RNA transcript (pre-mRNA) has to be processed before it can exit into cytoplasm where translation happens

In prokaryotes

mRNA can be translated by ribosomes while RNA polymerase is still making transcript

5' cap (Eukaryotes) how is it added and why is it needed?

1. GTP added 5’ → 5’ during transcription by capping enzyme attached to RNA Pol II

2. Guanasine cap is methylated by methyl transferase

3. required for efficient translation & stability

5' cap (Eukaryotes) what does it do?

protects RNA transcript from degradation & recruits translation machinery (translation wouldn’t happen without it)

3' poly A tail (Eukaryotes) Purpose:

Stabilize mRNA from exonucleases, Interacts with 5’ cap to position ribosome, Signal for export from nucleus into cytoplasm

3' poly A tail (Eukaryotes) - 2 step process

1. makes 3' end (cleavage of mRNA @11-30 bases downstream of AAUAAA) (signal for where cleavage happens before poly A tail added)

2. polyadenylation: 50-250 adenines added to 3’ end of RNA by poly A pol

Transcription Termination in Eukaryotes

Polyadenylation: causes transcription termination

complex protein machinery binds & cleaves DOWNSTREAM of signal, leaving a free 5’ end that binds Rat1

UPSTREAM: RNA becomes pre-mRNA that receives a poly-A tail; Rat1 (a 5’->3’ exonuclease) degrades tail to RNA pol II to stop transcription

mRNA Splicing

removing introns & pasting together exons

Exons:

sequences found in genomic DNA & mature mRNA (expressed regions) - protein coding sequences & untranslated regions

Introns:

sequences found in genomic DNA but not in mature mRNA (disappear) – interrupt coding region → Most Eukaryotic genes have many introns

Introns rely on

specific Consensus sequences indicate where the intron is (5’ splice site at beginning, 3’ splice site at end). Branch point A inside of intron (Adenine residue) helps to initiate the process of intron removal

Introns in simple terms

taken out of mRNA

Exons in simple terms

translated

snRNPs (small nuclear ribonucleoproteins)

RNA & protein

• recognize & bind at consensus sequence

• combine with other proteins ==> spliceosomes

result: spliced mRNA (lariat loop structure) in intron fragment → joins neighboring exons together

Between species, intron size varies highly. As eukaryotes evolve to greater complexity, genes evolve to include more introns. Prove that introns are important for regulatory roles. More introns:

increased opportunity for alternative splicing

Extreme Splicing example: human dystrophin gene has a huge amount of BP. splicing removes all noncoding introns. Goes from 2,500,000 nucleotides to 14,000 nucleotides

Alternative Splicing

diff pre-mRNAs can be spliced differently from same transcript

result: diff mature RNA → diff proteins with diff functions

(alternate proteins = isoforms)

Exon Skipping:

exon can either be included, or some can be excluded

Mutually Exclusive Exons:

only 1 of 2 alternatives is included in mature RNA

Alternative 5’ or 3’ splice sites:

splice sites changing, results in a change in boundary of exon (add or remove nucleotides)

Some animals can do Trans-splicing:

exons from separate RNA molecules join together

Mutations don't always occur in exons, but can affect introns as well. Mutations in introns may affect

intron splicing-can later cause disease (ex: hemophilia B in royal family)

Complex eukaryotes don’t have more genes, but they do have

more alternative splicing!

Translation

Converts nucleic acids (mRNA) to amino acids

The genetic code:

4 possible nucleotide bases but 20 possible amino acids

How is genetic code converted?

must be a specific translation from bases to each amino acid (aa); if 1 base per aa = 4 possible; if 2 bases per aa = 4 x 4 = 16 possible; if 3 bases per aa, then 4 x 4 x 4 = 64 possible combinations ( ”degenerate code” have more codons that encode 20 amino acids)

Nucleotides are “read” in

3 base non-overlapping units called codons

Translation: Why is identifying the template strand vs the non-template strand so important? On DNA, template strand is 5’-TAA-3’, nontemplate strand is 3’-ATT-5’; RNA from template strand is 3’-AUU-5’, nontemplate strand is 5’-UAA-3’ If you got the template strand wrong, it would code to different amino acids! So how are codons read?

Very important to read from 5’ → 3’

Impact of code degeneracy:

Multiple DNA sequences can encode the same protein

Codons are read as

non-overlapping units & ****Each strand has three reading frames

3 reading frames produce very different polypeptides from 1 gene. Which is “right”?

Always start with an AUG codon (Met) & end with a stop codon → likely (RF1 most likely!) proteins have ~50+ aa between AUG and stop; if its less than 50 aa its likely a shift took place

The nearly “Universal” Genetic Code

Many stops codons encode for amino acids (in Mitochondria)

Translation: What is the mechanism? Polypeptide synthesis: what is needed?

mRNA template, transfer RNAs (tRNAs) & amino acids, Ribosomes (ribosomal RNA & protein)

Transfer RNA (tRNA) – adapter (connect codons in mRNA)

Short single stranded (many modified bases so that it can have correct shape & do correct job)

Anticodon (bottom of tRNA) pairs with complementary mRNA codon, aa attached to 3‘ end

Amino acid attachment site is fixed! How is the correct a.a attached?

Aminoacyl-tRNA synthetase (diff for each aa)

• ENSURE: loads right aa onto right tRNA

• recognized by sites on ALL arms of tRNA

A tRNA with an amino acid attached is called a

charged tRNA (tRNA^XYZ)

Codon and Anticodon pairing: Wobble positions: We have 61 codons, but cell doesn’t need 61 different tRNAs because of wobble pairing →

3rd base of the mRNA(1rst base in anticodon) is going to pair less strictly with the first base of the tRNA anticodon. a single tRNA can recognize multiple mRNA codons → important for having fewer tRNAs

Ribosomes are the site of? Where are they located? complex structures composed of rRNA and protein has a….?

site of protein synthesis; cytoplasm or on ER (determines where proteins are going)

has a large subunit (50S- for catalyzing peptide bonds between amino acids) & small subunit (30S- for starting translation)

Translation Stages (bacterial):

Initiation, Elongation, Termination

Initiation of Translation in Prokaryotes

mRNA & 2 subunits of ribosome

1. small ribosomal subunit binds RNA near Shine-delgarno sequence (bacterial cells)

2. helps position ribosome by start codon (AUG) to start

3. Better match to shine sequence = more translation

Initiation Factors of Translation in Prokaryotes

1. IF1 +IF3 bind to small subunit(makes sure initiation occurs properly)

2. IF2 (bound by GTP) brings tRNA to start codon → binding of fMET tRNA for AUG codon

3. GTP hydrolyzed → large subunit (50s) comes in & binds (energy comes from IF2, bound by GTP)

Elongation of Translation in Prokaryotes 3 steps: (cyclic)

1. charged tRNA (w elongation factor proteins) to A site

2. peptide bond between amino acids in P & A sites

3. translocation of ribosome down mRNA (5' to 3') moves A → P site (tRNA shifts to P site, A site free)

Elongation factors (EF) of Translation in Prokaryotes

EF-Tu brings new tRNA to “A” site

EF-G moves ribosome down 1 codon

If there are secondary structures: Helicase working downstream of ribosome unwinds them in mRNA

Termination of Translation in Prokaryotes

when ribosome matches a “stop” codon bc no tRNA matches stop

Termination proteins: of Translation in Prokaryotes: release factors (RF) bind to

RF1: UAA & UAG

RF2: UGA & UAA

RF3: GTP dislodges complex (release ribosome: recycled so mRNA can be transcribed again!)

Premature termination can arise if mismatches are found in the

“P” position

Eukaryotic Translation Initiation

MANY more Initiation factors (eIF)

No Shine-Dalgarno → uses 5' cap & eIF1 to bind to a small ribosomal subunit (scans mRNA (5’ to 3’) for first AUG (start)

often in a consensus sequence (Kozak sequence 5'-ACGAUGG-3‘) to assemble rest of ribosome & start synthesis

****PolyA tail promotes translation in eukaryotes

****only 1 protein/mRNA

50S prokaryotes & 80S eukaryotes