TRANSCRIPTION *from book

PROKARYOTES
Two types of Enzymes
1. Constitutive
Synthesized constantly
2. Adaptive/Inducible
Synthesized at varying rates
Lac Operon
Synthesizes -galactosidase (Z+: hydrolysis of lactose to glucose + galactose),
galactoside permease (aka lactose permease; Y+: brings lactose into cell),
thiogalactoside transacetylase (Z+: in vitro adds acetyl group to the C6-OH of a -
galactoside)

Has a repressor attached to the operator (O) gene until lactose attaches to it and
it dissociates and synthesis of the genes happens

DNA Sequences
1. Cis-acting
Sequences that are on the same DNA molecules
2. Trans-acting
Sequences are on a different molecule

RNA Polymerase
* couples ribonucleoside triphosphates on DNA templates that driven by the release and subsequent
hydrolysis of PPi
* EUKARYOTIC 4 or 5 that synthesize different classes of RNA
* Holoenzyme: composed of 5 subunits however once RNA synthesis has been initiated the initiation
factor falls of; the 4 enzymes are then called the core enzyme
* Several functions
1. Template Binding
Holoenzyme binds to promoters; recognized by the initiation factor
Promoter sequences:
Pribnow box @ -10 TATAAT
-35 TTGACA
separated by 16 19 sequences
Mutations
Up
9 Increase rate of transcription
Down
9 Decrease rate of transcription
initiation nucleotide (+1), usually A or G (CAT or CGT sequence)
transcription bubble
2. RNA chain initiation
Bacterial RNA has a 5 triphosphate group
Does NOT require a primer
Abortive initiation
As synthesis goes on, if the RNAP doesnt let go of the promoter there is a
conformation tension that builds up, scrunching increase sized of the
transcription bubble occurs and the RNAP then lets go of the newly synthesized
RNA fragment to ease the stress
 Rifamycin B or Rifampicin mechanically inhibits chain elongation by blocking the location;
only on bacterial RNAP
3. Chain elongation
Occurs in the 5 3 direction
Causes supercoiling ahead (fixed by gyrase or topoisomerase II) and negative coiling
behind (fixed by topoisomerase I)
Antinomycin D tightly binds to duplex DNA and inhibits transcription and replication of
DNA
4. Chain Termination
Intrinsic or Spontaneous terminator
Induces termination without assistance
7 to 10 consecutive A-Ts with As on the template strand
G + C rich segmenf with palindromic sequences up stream of the A-Ts
Hairpin structure is made; RNA pullout model hairpin formation mechanically
pulls out RNA from the RNA-DNA structure
Rho dependent
Rho factor proteins stops synthesis

EUKARYOTES
RNA Polymerases
*small and large subunit
1. Pol I
Synthesizes precursors of rRNAs
2. Pol II
Synthesizes precursors of mRNAs
3. Pol III
Synthesizes precursors of 5S rRNA, tRNA

*from powerpoints
TRANSCRIPTION and RNA PROCESSING
Gene
1. Transcriptional region
Part of DNA mRNA
2. Regulatory region
3. Promoter
Prokaryotes: -10 (Pribnow box) and -35
Prokaryotic Transcription
Very little noncoding sequences
Usually organized into polycistronic operons
Clusters of >1 coding region with only 1 promoter
Ex: lac operon 1 promoter, 1 operation, 3 structural genes
Promoters:
-10 and -35
RNA Pol holoenzyme; 2 , 1 , 1 , 1
initiation factor that ensyre that RNA pol binds stably to DNA only at the specified
promoters, will otherwise destablize association
antibiotic inhibitors
Antinomycin D binds to DNA
Rifampisin binds to polymerase and mechanically prevents elongation
Eukaryotic Transcription
Has a lot of noncoding sequences
Introns and exons are present
Promoters
-25 (Hogness Box) and -75 (CAAT box)
3 Different Polymerases
 Pol I
5.8S, 18.5S, and 28S rRNA
Pol II
Protein coding genes, snoRNA, miRNA, siRNA, snRNA
Transcription Factors
9 TFIID
TBP recognizes TATA box
TAF - recognizes sequences near transcription start point; regulates
DNA- binding by TBP
9 TFIIB
Recognized BRE (TFIIB recognition element) elements
Accurately positions RNA pol
9 TFIIF
Stabilized RNA pol interaction with TBP and TFIIB
Attracts TFIIE and TFIIH
9 TFIIE
Attracts and regulates TFIIF
9 TFIIH
Unwinds DNA at start point
Phosphorylates SER5 of the RNA pol
Releases RNA pol from Promoter
Assembly of the PIC (Pre-initiation complex)
9 TBP to promotor recruits TFIIB to TFIID and TFIIA
9 RNA Pol II and TFIIF are bound together recruited by TFIIB
9 Pol II recruits TFIIe which then recruits TFIIH
9 TFIID TFIIA and TFIIB RNA Pol II w/ TFIIF TFIIE TFIIH
TATA box and Initiator (Inr) are required for transcription
9 Other elements needed: BRE and DPE (downstream promoter element), and
regulatory sequences
Accessory Proteins
9 Transcriptional activators binds to enhancers
Enhancers near in prokaryotes; either near or far from eukaryotes
9 Mediator
9 Chromatic-modifying enzymes
Chromatin remodeling complexes + histone-modifying enzymes
Pol III
tRNA, 5S rRNA,
Transcription Process
Termination
Pol III ends at T rich sequences
Pol I ends at a terminator site
Pol II downstream from the 3 end of mature transcript
9 Mature transcript includes a poly(A) tails
RNA Processing
Capping
9 Addition of a 7-methylguanylate to the 5 end
Polyadenylation
9 Add a polyA tail at 3 end
Binding sites for proteins that will protect mRNA from ribonucleases
Splicing
9 Remove introns
9 Join exons
9 Introns
Have a clear signal for slicing
Splice donor site GU
Branch site A
Py rich site
AG to Splice acceptor site
9 Spliceosome
5 small nuclear RNAS: U1, U2, U4, U5, U6 (NO U3 GAGA ANNA)
 U1 attaches to 5 splice site and U2 to branch site
U4, U5, U6 attaches to 5 splice site and cleaves it
5 end attached to A in branch site, U1 and U4 released
5 end of exon 2 is attached to 3 end of exon 1
intron is released
Mechanism
A @ branch attacks splice donor site, exon 1 attacks exon 2 +
splice acceptor site
RNA Editing
Base of a RNA molecule is altered by specific enzymes
Ribozymes
Catalytic RNA
RNA self-splicing
Does NOT use snRNAs
Two kinds
9 Group I
Requires G as cofactor
9 Group II
Does not require G as cofactor
A at branch point
RNA might have been the first biomolecule
Store genetic info despite unstable
Catalytic action

REGULATION OF GENE EXPRESSION

Prokaryotes
Constitutive expression
Transcribed continuously
Facultative
Transcribed only when needed
Gene regulation usually at transcriptional initiation
RNA Pol activity regulated by regulatory proteins:
Activators positively
Make decisions of the best sources for C, N, etc
Repressors negatively
Decides when synthesis of smth should occur
Interacts with operator sequence and affects promoter accessibility
Transcriptional Control
2 Mechanisms
Induction ON
9 Makes use of an inducer usually the substrate
Repression OFF
9 Makes use of a repressor usually attached at the operator
2 Kinds of genes
Inducible
9 Only transcribed once induces
Repressible
9 Continuously transcribed unless repressed
lac operon
inducible operon
1st presence or absence of lactose
2nd positive control by cAMP; positive regulator (activator)
Trp operon
Repressible operon
1st When Trp is available, it stops
2nd attenuation
Arabinose operon
Converts arabinose to usable form
 Both + and control
Synthesizes AraC which at high concentrations inhibits further transcription of the gene
Effectors
On Negative Regulation Repressors
Causes dissociation of repressors
9 Allows from transcription
Causes association of repressor
9 Prevents transcription
On Positive Regulation Activators
Dissociation of activators
9 Prevents transcription
Association of activator
9 Allows transcription

Eukaryotes
Types of Control in Eukaryotes
Transcriptional
RNA Processing
RNA Transport
Translation
Protein Activity
Transcription Factor: p53
Transcription factor and tumor suppressor protein
Regulates expression of Mdm2
Genes for growth arrest, DNA repair, and Apoptosis
Transcription Factors
Motif Structures
Zinc Finger
9 C2H2
CX2-4CHX2-4H
X is any amino acid
2 cys, 2 his, 1 Zn
9 C4
CX2CX13CX2CX14-15 | CX5CX9CX2C
2 Zn, 8 cys; 4Cys/Zn
Steroid hormone receptors
9 C6
CX2CX6CX5-6CX2CX6C
Yeasts Gal4
6 Cys reacts with 2 zinc ions
Helix-Turn-Helix
9 C terminal helix fits into DNA major groove
9 DNA binding proteins
Leucine zipper
9 Binds DNA as dimers
9 2 helixes one from each monomer, held together by hydrophobic
interactions
9 has a Asparagine that is highly conserved
Helix-Loop-Helix
9 Looks similar to a leucine zipper
9 Myoblast determination proteins
Activity is synergistic
Control of TFs
Protein synthesis
Ligand binding
Covalent modification
Addition of a second subunit
Unmasking
Stimulation of Nuclear entry
 Release from Membrane
Repressor Proteins
Competitive DNA binding
Masking activation surface
Direct interaction with general transcription factors
Co-activators and Co-repressors
Enhancers are stopped by Boundary/Insular elements
Recognized by several non-histone proteins

Study of Gene Regulatory Proteins

Gel Shift Arrays
Unbound DNA fragment; DNA fragments + protein/s; DNA + protein + antibody
Sp1 with GC box
Adv: sensitive
Dis: requires a stable complex, no info where protein binds
DNA footprinting
Nucleases with DNA-protein complexes
DNA is cleaved at portions not surrounded by protein
A footprint is generated
FOR: known protein and unknown DNA target
Reporter Gene Assays
Reporter gene is fused with GOI
Usually plasmids are used
Why that gene?
Products can be easily identified
Products are selectable markers
Cell Cultures
For in vitro analysis
Cells capable of proliferating indefinitely
Usually cancer cells
Maintaining
37C, 5% CO2
Cell growth media depends on cell type
Medium requirements
9 Bulk ions like Na K Cl Ca Mg P HCO3- and CO2
9 Trance elements like Fe, Zn, selenium
9 Glucose (sugar/C source)
9 13 essential AA
9 Vitamins
9 Choline, Inositol
9 Serum
9 Antibiotics to prevent bacterial or fungal contamination
Phenol red to indicate if pH is good for cell
Inverted microscope
Transfection
Introduce foreign DNA into eukaryotic cells
Creating holes in cell
DNA is not usually incorporated into cells genome
Is not reproduced
Transiently expressed
Goals
Expression of protein product
Analysis of effect of DNA transfected on cell function
Usually involves reporter gene assays
Common Reporter Genes
GFP green fluorescent protein glow green under UV
Luciferase enzyme reaction with luciferin light
lacZ enyme
9 Blue-White Screening
 9 Presence of X-gal which processed by lacZ
9 LacZ sequence has a MCS
9 If plasmid is successfully recombined with another DNA colony is white
9 Not recombined colony is blue
Cholamphenicol acetyltransferase gene
Yeast 2 hybrid system
Discovers protein-protein and protein-DNA interactions
Protein 1 DNA binding domain
Protein 2 transcription activation domain

TRANSCRIPTION AND CHROMATIN REMODELLING

Chromatin
DNA bound to both histone and non-histone proteins
Nucleosomes
beads on a string
histones positively charged AA
DNA negatively charges
Favorable interaction between histones and DNA
Between nucleosome core is a linker DNA with a histone H1
Repeat lengths can be measures using mircococcal nucleases
Silent and active chromatin show different patterns
Types
Euchromatin
Transcriptionally active; less compact
Heterochromatin
Transcriptionally LESS active; more compact
Structure Modification
Chromatin Remodeling Complex
Nucleosome sliding access to DNA
+ histone chaperone removes histones
+ histone chaperone histone variants that allow greater access
Makes use of ATP
Shifts nucleosome with respect to DNA; can expose or occlude regulatory
sequences
Mechanism
9 Nucleosome sliding
9 Remodeling of Nucleosome
9 Nucleosome Displacement
9 Nucleosome Replacement
Histone Modifying Enzyme
Modifies histones destabilizes compact form
Different patterns of covalent modification gives cell history
Modifications
9 Acetylation of lysine
Reduces affinity of histone and DNA
Regulated by
Histone Acetyltransferases enhances
Histone Deacteylaces represse
IMPORTANT: CBP/p300 interact with numerous
transcription regulators
9 Methylation of lysine
9 Phosphorylation of serine
Modification state
9 Trimethyl @ 9 gene silencing by heterochromatin formation
9 Trimethyl @ 4 & Acetylaiton @ 9 gene expression
9 Trimethyl @ 27 gene silencing
Does NOT alter nucleosome position
TRANSLATION
Sense/Coding stand highly similar to mRNA except T (DNA) U (RNA)
Antisense/Template
Translation
3 reading frames
Always start at AUG
Open reading frames
Long sequence without stop codons
Rare
Codes for a protein usually
Components
mRNA, tRNA
tRNA
9 D loop
9 Anticodon loop
9 T loop
9 AA bound to 3 end
Ribosomes
Not membrane bound
Prokaryotes
9 70S = 50S + 30S
Eukaryotes
9 80S = 60S + 40S
A ribozyme
9 3 tRNA binding sites:
A - aminoacyl
P - peptidyl
E exit
AA of tRNA at A position attacks AA at P position
Elongation and Release Factors
Initiation of Translation
PRO
ribosomes binds to specific sequences (8 nt long) upstream of AUG codon
Shine-Dalgarno series
EU
ribosome binds to 5 cap
Kozak sequences to select which AUG
Met-tRNA
2 types in PRO
fMet to start protein chains
Met Med residues
Termination
Release Factors
RF1 UAG and UAA
RF2 UGA and UAA
RF3 GTP binding protein; promotes cleavage of the peptidyl-tRNA
Inhibitors
On bacteria
Tetracylcine
Streoptomycin
Chloramphenicol
Erythromycin
Rifamycin
On bacteria and eukaryotes
Puromycin
9 Premature release of peptide chain
 Actinomycin D
9 Prevents RNA synthesis
On eukaryotes
Cycloheximide
Anisomycin
-amanitin
diphtheria toxin