eukaryotic transcription (ch. 8)

what is chromatin and what is its role

chromatin = DNA and protein (histone) complex

plays a role in gene regulation

RNA pol. 1 function

transcribes (3) rRNA genes

RNA pol. 2 function

transcribes mRNA’s, snRNA

siRNA, miRNA

RNA pol. 3 function

transcribes tRNA, (1) snRNA, (1) rRNA

siRNA, miRNA

what does RNA synthetase do

aminoacyl tRNA synthetase - primes tRNA with correct AAhw

what do eukaryotic (and archael) RNA polymerases share (and number of subunits)

they share 5 homologous subunits with bacterial RNA pol.

have an additional 6-11 subunits

eukaryotic promoter elements - what is contained in the promoter

TATA box (Goldberg-Hogness box) — -25 (most common, most consistent promoter consensus sequence)

CAAT box — -80

CG-rich box — -90

CAAT and CG-rich boxes are more variable

eukaryotic promoter elements have MORE VARIATION than in prokaryotes

what is the principle binding site during promoter recognition

TATA box

steps of eukaryotic promoter recognition (MEMORIZE)

TFIID (containing TATA-binding protein + TBP associated factor) binds to TATA box —> initial committed complex

TFIIB, TFIIF, and RNA pol. II join complex —> minimal initiation complex

TFIIE, TFIIH join the complex —> complete (preinitiation) initiation complex

complete complex directs RNA pol II to +1 position, RNA pol II is released from GTF’s, and begins to assemble mRNA

complete initiation complex contains multiple TF’s which are referred to as General Transcription Factors (GTF’s)

RNA pol I promoters & initiation

ribosomal genes found in the nucleolus (nucleolus contains rRNA and many copies of rRNA genes)

promoters contain two functional sequences near tcr start:

• core element (-45 to +20) — initiates transcription. bound by sigma-like factor 1 (SL1) protein

• upstream control element (-100 to -150) — increases efficiency/level of transcription. bound by upstream binding factor 1 (UBF1)

binding of UBF1 and SL1 bends DNA, RNA pol 1 is then recruited to the core element to initiate tcr. RNA pol 1 sits between -45 and +20, where SL1 is located

RNA pol III promoters & initiation

promoters have an internal control region (ICR)

ICR’s are contained within the transcribed region - contain 2 short sequences (box A & box B/C) separated by ~25 base pairs. +55 and +

TFIII proteins binds to two box regions, then RNA pol III binds upstream of TFIII’s (near +1 and box A) and begins transcription

• TFIIIA to box C, facilitates binding of TFIIIC to box A. TFIIIB binds to TFIIIA&C.

have some variation and upstream regulatory elements

which transcripts are more stable

eukaryotic

1. mRNA 5’ capping - when and how?

after first 20-30 nucleotides added to mrna (occurs during transcription)

enzyme: guanylyl transferase adds guanine to 5’ end of premRNA. unique 5’-5’ bond, connected by triphosphate bridge

guanine is methylated, and possibly nearby nucleotides.

name of cap: 7-methylguanosine

FUNCTIONS of 5’ capping

protect from rapid degradation, facilitate transport out of nucleus, facilitating intron splicing, enhance translation by orienting ribosome on mRNA

what are the three steps of 5’ capping

1. guanylyl transferase removes gamma (third) phosphate from the first nucleotide (2 left)

2. gamma and beta (first and second) phosphages are removed from guanine triphosphate

3. guanylyl transferase joins the guanine monophosphate to the 5’ end of mRNA, forming a 5’ to 5’ triphosphate linkage. guanine monophosphate is methylated

FUNCTIONS of polyadenylation

facilitate transport of mRNA out of nucleus, protect from degradation, enhance translation by enabling ribosomal recognition of mRNA

***histone genes do not undergo polyadenylation

what are the steps of polyadenylation

1. a) CPSF (cleavage and polyadenylate specify factor) binds near polyadenylation signal sequence (5’-AAUAAA3’). b) CstF binds to U-rich region. c) CFI & CFII & PAP (polyadenylate polymerase) also bind

2. pre-mRNA is cleaved 15-30 nucleotides downstream of the polyadenylation signal sequence (cleavage site). cleavage releases a fragment bound by CstF, CFI, and CFII that is degraded

3. CPSF and PAP add 20-200 adenines (mostly PAP) to the 3’ end

4. after first 10 A’s are added, PABII (poly-A binding proteins) join near PAP to increase rate of polyadenylation

what is located at the end of the transcript for mRNA (on DNA) in order from 5’-3’

stop codon, 3’UTR, polyadenylation signal sequence, cleavage site, U-rich region

torpedo model for transcription termination

**polyadenylation and termination are connected by torpedo RNase activity

(after 3’cleavage and poly-A, the transcript mRNA is still attached to RNA poll II)

RNase digests residual transcript (downstream of poly-A) like a “torpedo” aimed at residual mRNA that is still attached to RNA pol II

when RNase catches up to RNA pol II it triggers termination and releases pol from DNA

who discovered “split genes”

Roberts and Sharp

what is R-looping

technique in which DNA for a gene is isolated, denatured, and hybridized to mature mRNA from the same gene in order to identify introns. regions of DNA that are introns have no complementary region (they are single stranded) and make loops

what kind of introns undergo self splicing and how does it work?

group 1 introns: self splicing ribozymes that catalyze their own excision. work in some mRNA; and tRNA and rRNA precursors (in bacteria, simple eukaryotes, and plants). two trans-esterification reactions excise intron and ligate exon ends

group 2 introns: self splicing ribozymes found in archaea and bacteria, & in Eukaryotic mitochondria and chloroplast genes. form complex secondary structures, occurs in a lariat-like manner

what are the intron splicing signals for pre-mRNA

GU-AG rule

beginning of intron (5’ splice site) has GU

end of intron has 11 nucleotide consensus with a Py-rich region and AG (3’ splice site)

branch site is 20-40 nucleotides upstream of 3’ ending. Py-rich and contains branch point adenine (A) near the 3’ end

describe the splicing reaction for mRNA

snRNA protein called spliceosome removes introns (U1 binds 5’ splice site and U2 binds branch point A)

1. 5’ splice site is cleaved first and binds to branch point adenine, lariat intron structure is formed

2. 3’ splice site is cleaved, exon ends ligated

introns are removed one by one but not necessarily in order

describe the composition of the spliceosome

it is made up of snRNPs (small nuclear ribonucelomprotein particles). have subunits U1-U6. composition changes throughout process (U4 leaves to activate spliceosome)

how do the spliceosome components come together

SR proteins bind to ESEs (exotic splicing enhancers) which are sequences that are located on exons; SR proteins bind near the 5’ and 3’ splice sites. spliceosome components recognize the SR proteins and are able to bind & cleave in the correct area.

SR proteins also protect exons

what is the gene expression machine model?

carboxyl terminal domain (CTD) of RNA pol II functions as an assembly platform & regulator of pre-mRNA processing machinery

“Gene Expression Machine” = proteins that carry out capping, splicing, and polyA associate with CTD

CTD also contains torpedo RNase that digests residual mRNA and terminates transcription

how are alternate transcripts of single genes created?

there are 3 tcp-associated mechanisms: alternative pre-mRNA processing

1. mRNA can be spliced in alternative patterns/diff combinations

2. alternative promoters initiate transcription at diff start points

3. alternative sites for polyadenylation produce different mature mRNA’s

what percent of human genes undergo alternative splicing ? give an example

70%. less common in animals, rare in plants

CT/CGRP gene undergoes alternative splicing, based on different polyA sites

• thyroid cells use exon 4 polyA site —> calcitonin

• neuronal cells use exon 6 polyA site —> CGRP

drosophila Dscam gene

24 exons produce more than 38,000 diff polypeptides. each neuron gets its own gene (self/non-self recognition)

what controls alternative promoters of polyadenylation

variable expression of regulatory proteins in specific cell types

tRNA processing in EUKARYOTES

each tRNA gene is individually transcribed

rRNA processing in euk.

rRNA is transcribed in large precursor molecules, then cleaved into individual molecules by removal of spacer sequences by enzymatic cleavage. then rRNAs fold into more complex secondary structures, join ribosomal proteins, form ribosome subunits

why is there less tRNA’s than needed for all codon combinations

in MOST organisms - technically need 61, but usually only have 30-40 because of the third base wobble (third nucleotide codon match is variable)

some eukaryotic genomes contain all 61 tRNA genes, one for each codon

describe base substitution RNA editing (euk)

replace C with U

mammals, land plants, some single celled euk.

in humans - produce 2 apolipoprotein B proteins from one gene. base substitution creates early stop codon, creating two different lengths of apolipoprotein B protein in intestinal & liver cells