Gene Expression in Eukaryotes

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Gene Expression in Eukaryotes

mRNA Transcription and Processing


Eukaryotic Transcription Machinery• RNA polymerase II

– synthesizes all eukaryotic mRNA precursors

– composed of 12 different subunits

– remarkably conserved from yeast to mammals

• Polymerase II promoters– 5' side of each transcription unit (mostly)

– TATA box at 24-32 bases upstream from start site

– Consensus: 5'-TATAAA-3'

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.18a


Eukaryotic Transcription Machinery• Initiation of transcription

– Requires a number of general transcription factors (GTFs)

– Their precise roles remain to be determined

– General = conserved in a variety of genes and organisms

– A preinitiation complex assembles at the TATA box

• Required before Pol II binds

– First: TATA-binding protein (TBP)

• TBP has 10-stranded sheet curved into a

• saddle-shaped structure that sits astride the DNA

• TBP is subunit of TFIID (fraction D)


Eukaryotic Transcription Machinery• TBP binding distorts DNA conformation

– Bound DNA develops a distinct kink

– DNA duplex becomes unwound over ~8 bp

• TBP is a universal TF

– mediates binding of all 3 eukaryotic RNA polymerases;

– present in 3 different protein complexes

• TFIID (pol II)

• SL1 (pol I)

• TFIIIB (pol III)


GTF’s and initiation• 3 GTFs interact with promoter on DNA

– TBP of TFIID

– TFIIA

– TFIIB)

• provide platform for multisubunit polymerase + TFIIF


GTF’s and initiation• followed by another pair of GTFs

– TFIIE & TFIIH

– TFIIH is only GTF known to possess enzyme activities

• 2 of its subunits act as DNA helicases

• allows polymerase access to template strand

– Another TFIIH subunit functions as protein kinase

• phosphorylate RNA polymerase

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.19


Elongation– One GTF (TFIID) may be left behind at

promoter• Future initiation?

– Other GTF’s are released from the complex


(CTD) of largest polymerase II subunit

• 7 amino acid sequence (-Tyr-Ser-Pro-Thr-Ser-Pro-Ser-); • in mammals, 52 repeats • All but 2 prolines are targets for phosphorylation • preinitiation pol II is nonphosphorylated• when transcribing, it is heavily phosphorylated• phosphorylation is likely a trigger for transcription• An elongation complex

– a number of large accessory proteins– > 50 components – total molecular mass of >3 million daltons

• Probably, template moves through immobilized machine



Regulation• Specific transcription factors

– bind to other sequences (CAAT-box, GC-box, enhancers)

– activate (or prevent) preinitiation complex formation

– Determine polymerase initiation rate


mRNA properties• They are found in the cytoplasm

• They are attached to ribosomes when they are translated

• significant noncoding, nontranslated segments

– ~25% of each globin mRNA is noncoding

– Noncoding portions are found on both 5' & 3' ends

– have sequences with important regulatory roles

• altered ends not seen in prokaryotes

– 5' end - methylated guanosine cap

– 3' end - string of 50 - 250 adenosine residues [the poly(A) tail]

– histones are an exception


Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.22c


Split Genes• Philip Sharp et. al. (MIT)

• Richard Roberts, Louise Chow et. al. (Cold Spring Harbor, NY) – Adenovirus introns

• Alec Jeffreys & Richard Flavell (1977, U. of Amsterdam) – Exons - parts of gene that contribute to mature RNA

product

– -globin gene

– Introns - intervening sequences




Split Genes• Split genes found in simpler eukaryotes (yeast,

protists)– fewer in number & smaller in size than in plants & animals

– Introns are found in all types of genes (tRNAs, rRNAs, mRNAs)

• Must be removed from primary transcript to make mature mRNA

• Shirley Tilghman, Philip Leder, et al. (NIH) – – R-loop formation seen in EM

– determined relationship between 15S & 10S (mature) globin RNAs

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.25a & b


Split Genes– Ovalbumin (protein found in hen's eggs)

gene • 7 loops form; correspond to 7 introns

• ~3 times more sequence than 8 exons

• Individual exons in all genes are typically < 300 bases

• Individual introns typically between 1,000 & 100,000 bases

• Explains hnRNA length

– Type I collagen gene• >20 times the length of mature message

• contains >50 introns


Processing• Ribonucleoproteins convert transcript to mature mRNA• Addition of a 5' cap & a 3' poly(A) tail • Removal of any intervening introns• 5' methylguanosine cap forms very soon after RNA

synthesis begins– 5' end initially has triphosphate

– First enzyme produces diphosphate

– Then, GMP is added in inverted orientation

– 5'-5' triphosphate bridge

– Next, guanosine base is methylated at position 7

– ribose methylated at 2' position

– 5' end modifications occur very quickly


Processing– May serve several functions:

• prevents exonuclease digestion of mRNA 5' end

• aids in transport of mRNA out of nucleus

• important in initiation of mRNA translation


The poly(A) tail – ~15 bases downstream from AAUAAA

– a protein complex carries out processing at 3' end

• associated with the RNA polymerase

• Included is an endonuclease

• poly(A) polymerase adds ~250 adenosines

– protects the mRNA from premature degradation

– Poly(A) tail allows affinity chromatography purification




Splicing – Must be absolutely precise– single base error changes reading frame– conserved sequence found at exon-intron

junctions– Usually G/GU at 5' intron end (5' splice site) – Usually AG/G at 3' end (3' splice site)– ~1% of introns have AT & AC, respectively– processed by different spliceosome (U12

spliceosome)



Splicing • functional differences as yet undetected)

– has U12 snRNA instead of the U2 snRNA of the major spliceosome

– U12 spliceosomes

• absent from yeast & nematodes

• present in plants, insects & vertebrates

• regions adjacent to intron contain preferred nucleotides

• play big role in splice site recognition (exonic enhancers)

• mutation can block intron excision


Splicing• human thalassemia caused by

mutations in globin splice sites• RNA catalytic abilities led to

understanding of splicing mechanism• Thomas Cech et al. (1982, U. of

Colorado) – RNA catalysis in pre-rRNA

– ciliated protozoan Tetrahymena (ribozymes)


Two types of intron splicing mechanisms described

• Group I introns (Tetrahymena pre-rRNA)– most common in fungal/plant mitochondria, plant

chloroplasts, & in nuclear RNA of lower eukaryotes, like Tetrahymena

– variable sequence, but similar 3D structures


Two types of splicing– Group II introns

• also self-splicing

• seen in fungal mitochondria & plant chloroplasts

• structure very complex & different from Group I introns

• go through intermediate stage (lariat, like cowboy rope)

– First step is cleavage of 5' splice site

– followed by formation of lariat

– covalent bond between intron 5' end & A near 3' end

– 3' splice site cleavage releases lariat

– allows exon cut ends to be ligated



Two types of splicing– Animal pre-mRNAs are processed like Group

II introns • difference is that intron cannot splice itself

• needs snRNAs & their associated proteins


hnRNA’s• hnRNP’s facilitate processing reactions

– spliceosomes remove introns• have a variety of proteins & snRNPs

• assembled they bind to the pre-mRNA

– snRNPs help remove introns from transcript


hnRNP’s• snRNPs required:

– U1 snRNP, U2 snRNP, U5 snRNP & U4/U6 snRNP (U4 & U6 snRNAs bound together)

– U6 is most likely to act as a ribozyme

– makes both cuts in the pre-mRNA required for intron removal




snRNPs: a dozen or more proteins• One family, the Sm proteins are present

in all of the snRNPs• they bind to one another & to a

conserved site on each snRNA• forms the core of the snRNP• Sm are targets of autoimmune

antibodies – systemic lupus erythematosus


snRNP’s• The other proteins of the snRNPs are unique to each

particle

– ATP-consuming, RNA helicases (unwind double-stranded RNAs)

– helicases are found within snRNPs

– at least 8 implicated in the splicing of yeast pre-mRNAs

• snRNAs are catalytically active (not the proteins)

– similar to group II introns, which splice themselves

– snRNAs closely resemble parts of the group II introns


snRNP’s• The proteins likely serve supplementary

roles– Maintaining the proper 3D structure of the

snRNA

– Driving changes in snRNA conformation

– Transporting spliced mRNAs to the nuclear envelope

– Selecting splice sites to be used


snRNP’s• snRNP proteins - not alone in mRNA processing

– SR proteins: large number of SR dipeptides

– thought to form interlacing networks that span intron/exon borders

• They help recruit snRNPs to the splice sites

• SR proteins have positive charge

• may also bind electrostatically to negatively charged phosphate

• assembly of splicing machinery occurs during RNA synthesis

• most of RNA processing machinery travels with polymerase



snRNP’s– Most genes contain a number of introns

– splicing reactions occur repeatedly on single 1° transcript

• introns may be removed in preferred order

• generates specific processing intermediates whose size lies between

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Gene Expression in Eukaryotes