Unit 3 Biology

Gene Expression in Prokaryotic Cells

Circular chromosome; occurs at the same time

RNA vs DNA

Single stranded, contains a ribose instead of a deoxyribose, and can contain more complex structures

rRNTp vs dNTP

Differences: 2’ —OH vs 2’ —H

Similarities: 3 Phosphates, added to 3’ -OH

Types of Prokaryotic RNA

mRNA and ncRNA

mRNA (how long?)

Messanger RNA; Long (average 3000 nucleotides), codes for proteins. Coding region is 1200 BPs

Contains a promoter sequence, regulatory sequence, coding sequence, and transcription terminator

Types of sequences in mRNA

Promoter: Determines where transcription starts

Regulatory Sequence: Determines when, where, how much mRNA is made

Coding Sequence: ORF, opening region frame

Transcription Terminator: Where transcription ends

ncRNA

Includes tRNA (transfer RNA), rRNA (ribosomal RNA), and sRNA (small RNA)

Gene

Unit of heredity, carries the genetic information for a protein (polypeptide) or RNA

Opening Reading Frame

Stretches of DNA that lack a stop codon; every 3 nucleotides encode for amino acid during translation of mRNA to form a proteins; there are 3 reading frames possible per DNA strand

Basic Structure of a Bacterial Protein-Coding Gene (DRAW)

Promoter: From -10 to -35 site, where RNA polymerase binds. Determines transcription start

Transcription Start Site (TSS): From +1

Opening Reading Frame (ORF): Protein coding sequence

Start and Stop Codons

Start; ATG

Stop: TAG, TGG, TGA

Template vs Coding Strand

Template strand is use for mRNA synthesis

Coding strand is identical to mRNA strand, except A is replaced with U

Transcription Overview

RNA binds to single stranded DNA template at the promoter

DNA helix unwinds

RNA is synthesized from rNTPS from 5’ to 3’ direction

5’ end of transcript is displaced from template as polymerase moves

How to know which strand is template strand for a gene? Draw

3’ to 5’

RNA vs DNA Polymerase

Doesn’t require a primer, only one nucleotide polymer is built, no proofreading

How many total reading frames are possibe?

6

Operons

Are in prokaryotic cells. Multiple ORF in one sequence, each has its own ATG and STOP sequence.

RNA Polymerase Structure

6 subunits, one leaves

Initiation

Consensus sequence upstream of TSS in the promoter

Sigma subunits binds to the -10 and -35 sequences to correctly position the enzyme

After initation, the sigma subunit disassociate and RNA polymerase can continue elongation

Elongation

RNA polymerase synthesizes. 17 bp are unwound in the transcription bubble. Moves at 50 nucleotides per second and the error rate is 10^4 or 10^5

Intrinsic Termination

G-C rich stem-loop structure of mRNA followed by a rich string of As. RNA polymerase dissasociates when it encounters the G-C loop.

Rho Dependent Termination

Rho binds to rut sequence upstream of termination site and acts as helicase to pause the RNA-DNA hybrid and facilitates the release of mRNA

Helix Turn Helix

Recognition of specific binding sites

Activator

Mediate positive regulation and bind to DNA

Repressors

Mediate negative regulation and bind to operators, block -10 part of consensus sequence

Inducers

Change the conformation of activators and repressors

Activator: Requires inducer to bind to active site

Repressor: Leaves operator in presence of inducer

LacZ

B-galactosidase enzyme that breaks down into glucose + galactose

LacY

Permease; permits entry of lactose into cell

LacA (don’t need to know)

Transacetylase

I component of Lac Operon

Creates repressor

Glucose and cAMP relationship

Low glucose leads to high levels of cAMP

Highest Transcription Levels

High lactose and low glucose

CAP

cAMP binds to CAP, an activator protein. CAP protein recruits RNA polymerase by binding to the C-terminal domain of the alpha subunit

Basal Expression

No repression and no activator

Lac Repressor Binding

Binds to DNA as a dimer, is symmetrical, helix-turn-helix

Jacob and Monod: Cis

Position and sequence are both important, DNA binding sites

Ex: Promoter

Trans

The protein made from the gene sequence is important, can diffuse

Ex: Regulatory proteins —> LacI acts in trans since it can be generated on the plasmid and still express wild type levels even if it can’t be generated on the chrommosome

Partial Diploid

Adding gene to the bacteria via plasmids; one chromosomal DNA and one bacterial DNA

Genetic Code Redundancy

• Triplet Code: 64 different 3 nucleotide “codons”

• Not 64 tRNAs

• Ex: For alanine there are 4 codons but not 4 tRNAs
  

tRNA

• Reads base pairs on mRNA

• Small 73-93 nucleotides

• Presence of modified bases such as inosine (I) and pseudouridine

• Contains STEM loops

• Amino acid attachment to 3’ CCA STEM

Aminoacyl-tRNA syntheases Draw

Charge tRNAs with specific amino acids

1) ATP and specific amino acid binds to tRNA

2) Amino acid specific tRNA binds to the syntheases

3) Recognizes the anticodon loop and binds to the amino acid (hydrogen bonding, van der Waals, hyrophoic)

4) Is released

Inosine

Deamination product of A, base-pairs with A, U, or C in the third (or wobble) position, on 5’ end of anticodon

Aminoacyl t-RNA linkage

Carboxyl group of amino acid is linked to 3’ OH of tRNA

Aminoacyl t-RNA

Intermediates in protein synthesis, not very stable, protected by EF-TU translation factor

Ribosome

Bind mRNA and tRNA

3 Binding Sites of mRNA

A: Aminoacyl/Acceptor, new tRNA molecule comes in
P: Peptidyl, Growing peptide chain
E: Exit, Site where tRNA will leave

Direction of mRNA and Amino Acid Chain

mRNA is read 5’ —> 3’, amino acid chain is addd to the COO- end

Ribosome Composition

Large Subunit: 50s = 23S rRNA +5sRNA +31 proteins

Small Subunit: 30s = 16S rRNA +21 proteins

Need to know: Made of RNA and protein, protein is more structural

IF2: Initiation of Translation in Prokaryotes

1) mRNA binds to small 30S ribosomal subunit

2) Initiation Factor 2 (IF2) delivers a special tRNA charged with formylmethionine (fMET) to the P site (AUG)

3) Once tRNA-fMet is in place, the 50S subunit of ribosome can bind, GtP bound to IF2 is hydrolyzed and released

Shine-Dalgarno Sequence

Ribosome binding site on mRNA that 16s rRNA binds to. Positions the mRNA at the correct spot so that AUG is at the P site

Formyl Groups

Blocks the reactive N atom of the amine group to help with stability and folding (not used in eukaryotes)

G protein Translation Factors

IF2, EF-TU and EF-G

Ef-TU

GTP-bound forms a complexed with charged tRNA = tRNA-AA(amino acid), if codon/anticodon interaction between tRNA and A site is correct, GTP on EF-Tu is hydrolyzed and Ef-Tu is released (A site conformational change)

Peptide Bond Formation

Amino acid end of tRNAs in P and A sites are in close proximity and a peptide bond is formed, catalyzed by rRNA

Between C//O in the amino acid on tRNA at the P site and N on the amino acid in the A site

EF-G

Hydrolyzes GTP to GDP for translocation of ribosome toward 3’ end

Ribozyme

rRNA catalyzes peptide bond, not protein

Termination

Mediated by Release Factor proteins that interact with stop codons in the A site

Molecular mimicry

Release factor mimics tRNA molecule

Polysome

Couple transcription and translation in prokaryotes

Transcription in Eukaryotes

1) Separation of transcription and translation in eukaryotes with

2) More complex transcription regulation.

• Three RNA polymerases

• General Transcription Factors GTFs

• cis acting elements are more varied

3) Extensive processing of mRNA

Nucleosomes (+Histone Types)

DNA (approx 160BP) is wrapped around a protein core of 8 histone molecules (4 different types H2A, H2B, H3, H4)

Levels of Chromatin Packing

DNA —> Nucleosome (10:1) —> Chromatin(50:1) —> Scaffold-associated chromatin (250:1)—> Condensed heterochromatin (5000:1)—> Compacted chromosome (8000:1)

Histone Structure

Histones interact with DNA minor groove; A/T base pairs are favored. Differ in N terminus tails

N-terminal tail

Targets of extensive modification such as acetylation and methylation (mostly at amino acids arginine/R and lysine)

Protein Classes and Histone Modifications

Writers: Covalently modify histone amino acids. Ex: Methyl transferases, acetyl transferases

Erasers: Restore modified to unmodified. Ex: Demthylases, deacetylases

Readers: Bind modified histone amino acids. Ex: Methyl readers and acetyl readers

Mammalian Gene Structure

Introns (leave) and Exons (stay)

Promoter: -25 TATA box, -200 nearby elements.

Enhancers: Distal (-10kb), proximal (-500kb), and downstream

Chromatin vs Non Chromatin DNA

Promoter Core: TATA and TSS Start site. Sequence needed for general transcription factors and RNA Polymerase to non-chromatin DNA

Promoter: Core promoter plus nearby elements. Minimal sequence required to recruit RNA polymerase to TSS in a cell (DNA packaged into DNA)

Enhancers

Work from far away to increase transcription, often involved in cell-type specific expression

Bacteria vs Eukaryotes

Bacteria: Ground state is on and promoters are DNA

Eukaryotes; Ground state is off and promoters are really protein-DNA complexes embedded in a chromatin environment

General Transcription Factors (GTFs)

GTFs and RNA polymerase assemble at the core promoter to form a Pre-Initiation Complex (PIC)
Need to Know: TFIIB, TFIID, TFIIH, pol II

Assembly of Pre-Initiation Complex

1) TATA-box Binding Protein (TBP) as part of TFIID; the anchor

2) TFIIB

3) RNA Polymerase II (binds to TFIIB)

4) TFIIH

TFIIH

1) Acts as a helicase

2) Phosphorylation of the carboxy terminal (CTD) RNA POLY II by a TFIIH kinase

Histone lysine acetylation

Acetylation neutralizes the positive charge of DNA, loosening the interaction between negatively charged DNA

3 Elements Required for In Vivo Transcription

1) Chromatin Remodeler to Reposition Nucleosomes

2) Formation of transcription initiation complex

3) Complexes that mediate these interactions

Nucleosome Remodeling

Gene activator protein binds to TATA box, bringing in histone acetylase to loosen the structure and chromatin remodeling complex to move histones

Chromatin Remodeler

Requires ATP

Mediator

1) Is required for transcription of most genes in cells

2) Interacts with RNA Polymerase, other GTFs and activator proteins bound to far-away enhancers

Activators and Repressors (Eukaryotic)

Bind to enhancers or nearby elements

Activators

two domains: DNA binding and activation domain. Targets of activation domains are recruited to the promoter by protein-protein interactions

Co-Activators

1) General transcription factors

2) Histone Code Writers

3) Chromatin remodoling complexes

Yeast Case Study

GAL 7, GAL 10, and GAL 1 are all coordinately regulated by a transcriptional activator Gal4.

Yeast Case Study: ± galactose

In the absence of galactose Gal80 binds to Gal4. In the presence of galactose, galactose binds to Gal3 which binds to Gal80 in the cytoplasm. This prevents Gal80 from getting into the nucleus, Gal 4 activation domain, binds a histone acetyltransferase coactivator complex (SAGA) and a mediator which recruit GTFs and RNA promoter.

Combinations of Activators

Different genes require different combinations of activators, can work cooperatively. Activation of the same gene at different times and places also involves different combinations of activators

DNA Looping

Transcriptional regulator proteins that bind to far-away enhancers are brought into the proximity of the promoter by DNA looping and interactions with the Meidator

C-Terminal Domain

CTD provides a binding site for factors involved in processing the transcript and regulating the activity of transcribed chromatin

Histone Deacetylases

Repressors can recruit co-repressors with histone deacetylase (HDAC) activity, similarly to how co-activators regruit Histone Acetyltransferase (HAT)

Mig1

Glucose availability induces the binding of a repressor called Mig 1 (galactose preferred source). Mig1 binds to repressor and recruits a co-repressor, HDAC

Heterochromatin

Constitutive heterochromatin form at all telomeres and sequences that flanked centromeres.

Fly Eye Experiment

White gene with no mutations: red eye color

White gene with mutations: white eye
Chromatin breaks off and rearranges so that gene is near heterochromatin which doesn’t get transcribed

HP1

HP1 is a protein that binds to methylated histone tails and recruits a histone methyltransferase

Barrier

Between heterochromatin and euchromatin, competing between whether they are methylated or acetylated

X Chromosome

Two versions of a gene on each chromosome, choice of X chromosome of X for inactivation occurs early in development

Genetics

DNA changed but transcription still occurs: ex mutation

Epigenetics

DNA unchanged but transcription doesn’t occur

Epigenetics

DNA unchanged but transcription doesn’t occur

Chromatin Modifications Associated with Heterochromatin and Epigenetic Silencing

Methylation of H3K9 (lysine at position 9) by HMT

Methylation at C5 of Cytosines by DNMT

mRNA processing

1) mRNA capping

2) Cleavage and polyadenylation (“poly A”) addition to generate the 3’ end

3) Intron splicing (removal of intron sequences)

Where is mRNA processed

Nucleus

5’ mrNA cap

RNA Pol II has a “stall factor” shortly after initiation to add the 5’ cap

1) Removal of the terminal Pi from the 5’ end

2) A guanine cap is attached to the 5; end using GTP (5’ to 5’ linkage)

3) Methylation of guanine