Fadool Exam 4

Promoter

Upstream of transcription start site

Can activate or repress transcription

Prokaryotic RNA polymerase

Synthesizes all prokaryotic RNA

σ subunit of RNA polymerase

Required for initiation of transcription.

Recognizes consensus sequences.

Multiple types of σ factors give specificity to gene expression and lead to coordination of gene expression.

Released after elongation begins.

Cis elements

DNA sequences that regulate genes on their same strand.

Trans elements

Proteins or functional RNAs that act on DNA to regulate transcription

TATA Box

Promoter sequence at -10 bp that aids in specificity of RNA pol binding and elongation

-35 consensus sequence

Prokaryotic promoter that aids in specificity of RNA pol binding and elongation

Transcription Factors

Proteins encoded by other genes that affect transcription

Topoisomerase 1

Causes single strand break to relieve (-) supercoiling.

Does not use ATP, but slow at relieving tension.

Topoisomerase 2

Double stranded break in DNA to relieve (+) supercoiling

Requires ATP and fast at relieving tension

Polycistronic messages

mRNA with multiple start codons that can encode multiple proteins (prokaryotes)

Operon model

Functionally related genes encoded together under a single promoter. Produces polycistronic mRNA.

Rho-independent termination

Intrinsic DNA sequence of inverted repeats transcribed into 3’ end of RNA. Results in RNA hairpin structure that disrupts RNA pol. processivity.

Rho-dependent termination

Intrinsic DNA sequences coded into RNA that are recognized by Rho protein hexamer. Rho protein translocates 5’ to 3’ to reach RNA pol complex. Breaks H bonds between RNA and DNA template. Destabilizes pol. and terminates transcription.

TATA Box (Goldberg-Hosness box)

Eukaryotic transcription promoter

Enhancer elements

Cis-regulatory elements that can be up to 100 kbp upstream or downstream of transcription start site.

Bind transcription factors and fine tune expression.

DNA loops so enhancers are spatially close to transcription start site.

Chromatin Immuno Precipitation (ChIP)

Fix RNA pol. II activity with formaldehyde.

Isolate nuclei and chromatin with centrifugation.

Chop DNA with endonuclease.

Immunoprecipiate with anti-pol. II antibody to identify RNA pol. II associated sites

TFIID

Large transcription factor complex, including TATA Binding Protein (TBP), that recognizes promoter regions and promotes pre-initiation complex assembly.

Nucleosome effect on transcription

Nucleosome = octamer of histones + DNA

Increased density of nucleosomes = inhibition of active transcription

mRNA cap

7 methylguanosine on 5’ end

Functions for translation initiation, increases stability, and influences splicing

Poly(A) tail

Non-Encoded adenine repeat at 3’ end

poly(A) consensus signal is 11-30 bp upstream and determines point of cleavage

Internal mRNA modification

m6A methylation influences splicing, degradation, and translation

Branch point

Single adenine nucleotide which provides binding site for 5’ end of interon

Lariat

Looping structure of intron following 5’ end binding to branch point

Spliceosome

5 RNA molecules and 300 proteins

snRNPs

snRNAs associated with proteins that are used in the spliceosome

1st step of splicing

pre-mRNA is cut at 5’ splice site, thus freeing exon 1 from the intron

The 5’ splice site binds to the branch point

2nd step of splicing

pre-mRNA is cut at 3’ splice site, thus freeing exon 2 from the intron

exon 1 and exon 2 are ligated, forming a spliced mRNA

Lariat is broken by lariat-debranching enzyme and intron is degraded by enzymes

Wobble hypothesis

3rd base of codon is less specific and may vary for tRNA binding

Aminoacyl tRNA synthetase

Charge tRNAs with appropriate amino acid by recognizing unique tRNA structure

Responsible for specificity of codons and tRNA

20 synthetases, 1 for each amino acid.

Start codon

AUG

Codes for N-formylmethionine (fMet) in prokaryotes

Codes for methionine in eukaryotes

30s ribosomal subunit (40s in eukaryotes)

Small ribosomal subunit

First subunit of ribosome to bind mRNA

Initiation Factor 3

Prevents binding of large subunit until small subunit has binded to mRNA

Initiation Factor 1

Protein required for the initiation of translation in bacterial cells; enhances the dissociation of the large and small ribosomal subunits.

Initiation Factor 2

Protein required for the initiation of translation in bacterial cells; forms a complex with GTP and the charged initiator tRNA and then delivers the charged tRNA to the initiation complex.

Shine-Dalgarno Sequence

Prokaryotic consensus sequence that is complementary to 16S rRNA (part of small ribosomal subunit) and positions binding of small ribosomal subunit at start codon.

Eukaryotic initiation

Small ribosomal subunit binds to 5’ cap and scans for AUG codon

Kozak consensus sequence

Eukaryotic consensus sequence around start codon that enhances ribosome scanning of start codon.

A site in ribosome

Where incoming charged tRNA enters the ribosome and pairs with the codon

P site in ribosome

Holds tRNA that carries the growing peptide chain and is where peptide bond formation occurs

E site in ribosome

Exit site where uncharged tRNA is extruded

Peptidal transferase

Catalyzes formation of peptide bond between previous carboxyl group and incoming amino group

Translation termination

GTP severs link between peptide and tRNA in P site

Polysomes

Multiple ribosomal complexes on a single mRNA

Streptomyocin

Alters pairing of charged tRNA to its codon, causing mRNA misreading

Aminoglycosides

Block initiation by preventing binding of fMet to the future P site

Chloramphenicol

Structurally looks like peptide bonds so is a competitive inhibitor of peptidal-transferase

Inhibition of peptide bond formation

Erythomyocin

Binds 50s particle and inhibits translocation by blocking peptide exit from export tunnel

Puromycin

Enters A site and terminates peptide chain

Tetrocyclines

Block charged tRNA entry into A site

Cis revertant

Additional frameshift that reverts reading frame back to wildtype

Trans revertants

Change in tRNA anticodon sequence that revert point mutations

Low dose X rays

Base changes (indels)

High dose X rays

Chromosomal rearrangements

Intercalation

Chemicals like proflavine may slip between bases, causing a frameshift

Transposions

Jumping genes

Photolyase

photo-activated repair enzyme in prokaryotes that captures blue light to repair UV-damaged thymine dimers in DNA

Glycosylase

Repair enzyme that cleaves bond between base and deoxyribose at damaged site, leaving sugar without a base.

AP endonuclease

Nicks upstream of debased site from glycosylase. DNA pol 1 or beta repairs sequence with 5’-3’ exonuclease activity. New nick at 3’ end repaired by ligase.

(Base excision repair)

Nucleotide excision repair

Helicase binds both ends of damage, thus unwrapping DNA as SSBP’s bind nondamaged ssDNA. Endonucleases cut out damaged DNA. DNA pol 1 and beta fill gap. Ligase seals 3’ nick.

SOS response in prokaryotes

Extreme amounts of DNA damage on both strands. Gene expression for specialized polymerases that can repair through DNA lesions (translesion polymerases)

Xeroderma Pigmentosa

Increased frequency of skin cancer associated with seven genes that affect DNA repair. Discovered through somatic cell hybridization.