BIO 230 Topic 3 - Transcription and Topic 4 - Translation

Location of transcription in bacteria

all in the cytoplasm

Transcription start site for bacteria

10 bp downstream of core promoter at the TSS (transcription start site)

transcription start site for eukaryotes

25-30 bps downstream of core promoter at the TSS (transcription start site)

transcription location in eukaryotes

nucleus, processing in nucleus and cytoplasm

enzyme transcribing genes

RNA polymerase II and makes complex with TATA binding protein

ALX1 gene

controls facial development for Darwin’s finches and is turned on or off by transcription factors

Promoter Region

regulatory DNA surrounding transcription start site (TSS) which helps recruit and regulate transcription machinery

Core promoter

6-10 bp DNA more immediately adjacent/upstream of TSS where general transcription machinery assembles, can be referred to as TATA box

Promoter-proximal regulatory elements

regulatory sequences near core promoter where sequence specific transcription factors bind

Order of binding to TATA box in transcription initiation

1. TATA Binding Protein (TBP) subunit binds

2. TBP recruits the rest of RNA polymerase II and other transcription factors which makes pre-initiation complex (PIC)

RNA Polymerase II

Directly transcribes DNA to RNA and operates from TSS to terminator sequence

Untranslated Regions (UTR)

5’-UTR is distance between TSS and ATG codon which marks start of translation for genes encoding proteins, these parts of genome are transcribed into RNA but not translated, can be 30-1000 bp long

Transcription factor binding site

cis-regulatory module, where sequence dependent binding happens, and most genes regulated by multiple transcription factors

Mediator complex

protein complex that determines interaction between transcription factor and pre initiation complex

Reason CRM is Cis-regulatory

physical stretch of DNA on exact same chromosome as gene it regulates,

why transcription factors are trans-regulatory

proteins that are produced elsewhere and must diffuse through cell to reach target DNA

Transcription factors consensus binding sequences

bind sequences 6-12 bp long, can be variable for different genes those some bases more frequent than others

Reporter genes

used to study gene regulation, coding region is replaced or supplemented with a reporter protein allowing time and location of expression to be easily visualized.

hox genes

master regulator genes where each gene encodes a transcription factor expressed in certain regions of the body,

hox proteins

transcription factors that regulate gene expression of developmental genes, errors in the timing of gene expression in hox genes leads to abnormal development

Two parts of transcription factors

one part interacts with pre-initiation complex called trans-interaction domain or activation domain (activating transcription) and other part binds to consensus sequence of DNA

cooperative binding transcription

binding of one TF makes it likely another TF will bind

structure of chromatin

DNA is wrapped around core histones to form nucleosome (10-nm fibre) and nucleosomes are linked to make 30-nm fiber.

nucleosome contents

146 base pairs of DNA wrapped around, composed of eight histone molecules with two copies of H2A, H2B, H3 and H4

chromatin fibers

nucleosome particles coiled into 30nm fiber which becomes solenoid chromatin fiber

open vs closed chromatin

closed chromatin is refractory to TF binding while open is available to TF binding

Pioneer transcription factors

can bind even when chromatin is closed, examples of cooperative binding

structure of nucleosome

all four core proteins have a “tail” of amino acids that sticks out away from DNA molecule that interact with other proteins and can be covalently modified

role of mediator complex

chromatin is looped to bring the transcription factors into interactions with pre-initiation complex

how does elongation occur

by adding one base at a time to 3’ end, using template strand of DNA as the guide meaning RNA has same sequence as coding strand

Rho dependent transcription termination in bacteria

helicase protein know as Rho binds to transcript and interacts with rut sequence downstream of stop codon, disassociating transcript from DNA

Rho independent transcription termination in bacteria

transcript has inverted repeat structure downstream of stop codon and when reapeats form hairpin structure through intra-strand base pairing, transcription terminates

Eukaryotes Transcription termination with RNAP 1 (ribosomal RNA genes)

RNA helices protein with some functional similar to Rho protein appears to be involved

Eukaryotes Transcription termination with RNAP 2

extends hundred or thousands of bases beyond end of coding region of the gene, two factors associated with C-terminal domain recognize poly-adenilation signal in transcript and recruit other proteins to continue and complete RNA processing.

CPSF (Cleavage and Polyadenylation Specificity Factor)

large protein complex that recognizes specific sequences of RNA, and once recognize Poly-A-Signal move from RNAP 2 to mRNA

CstF (Cleavage Simulation factor)

works alongside CPSF to help stabilize complex, once recognize Poly-A-Signal move from RNAP 2 to mRNA

Poly-A Signal (PAS)

specific sequence in pre-mRNA (usually AAUAAA) acts as stop sign

Xrn2 (Rat 1 in yeast)

a 5’ to 3’ exonuclease which binds to uncapped 5’ end of remaining RNA and rapidly degrades RNA in 5’ to 3’ direction effectively “chasing” RNA polymerase along DNA, then stabilizes polymerase causing it to release from DNA template

modifications to mRNA after transcription in eukaryotes

1. addition of 7-methylguanine as cap at 5’ end

2. addition of polyA tail at 3’ end

3. introns removed and exons spliced together

alternative splicing

many transcripts have more than 1 pattern of splicing, alternative splices occur within a cell or in cells of different tissues or at different times

types of alternative splicing

1. Exon skipping - exon 2 is included in some but not all copies of transcript

2. alternative 5’ or 3’ site - sequence at ends of exon 1 or exon 2 are included in some copies of transcript

3. mutually exclusive exons - either exon 2 or exon 3 is included but not both

how biochemically are introns spliced

1. group of five snRNP’s (small nuclear riobonucleoproteins) form a splicosome complex

2. snRNP’s bind to intron and cause it to fold in bringing 5’ to 3’ end of intron together and making a loop

3. intron detaches and splice sites connect to make a mature mRNA, most introns used in other processes, snRNP’s detach from intron and are used for splicing

aminoacyl synthetase

enzyme that loads or charges a specific amino acid to 3’ end of tRNA based on anticodon

structure of tRNA

stable single-stranded RNA with characteristic stems and loops generated by base pairing within the strand, 3d structure means one section of sequence sticks out as exposed bases, charged when amino acid attached to 3’ end

ribosome structure

composed of large and small subunit, including rRNA molecules and proteins and has 3 docking sites: A-aminoacyl site,P- peptidyl site, E- exit site

translation initiation

AUG Initiator codon brought to P site on ribosome by proteins known as initiation factors

translation elongation

at A site codon on mRNA is presented with anticodon on tRNA,