Chapter 3Gene Function

• TranscriptionProkaroyotesEukaryotes

• Transcript processing• Proteins• Translation• Genetic nomenclature

Transcription

RNA composition

ATP, GTP, UTP, CTP are substrates for RNA polymerase.RNA is a ribonucleoside monophosphate polymer.

RNA types

Informational:mRNA, encode amino acids, ~ 1% of total cellular RNAFunctional:tRNA, translationrRNA, ribosome components; 5S, 5.8S, 18S, 28S; bulksnRNA, splicingsiRNA, miRNA inhibitory (regulatory) RNAother small stable RNAs, signal recognition particle, RNaseP

ribozymes

Tandemly transcribedrRNA genes (NO).

Promoter codogenic strand

template strand

.....ApGpCpGpT......

.....TpCpGpCpA......

pppApGpCpGpU......

DNA5'

5' 3'

3'

mRNA

Transcription of a gene

5' 3'

Both DNA strands can serve as template for transcription

RNA polymerisation

The RNA strand grows from 5' to 3'.

Comparison of eukaryotic andprocaryotic RNA polymerases.

Companion site for Biotechnology. by ClarkCopyright © 2009 by Academic Press. All rights reserved.

RNA Polymerase Synthesizes RNA at the Transcription BubbleRNA polymerase is a complex enzyme with two grooves. The first groove holds a single strand ofDNA, and the second groove holds the growing RNA. RNA polymerase travels down the DNA, addingribonucleotides that complement each of the bases on the DNA template strand.

There are three eukaryotic nuclear RNA polymerases

5S rRNA, tRNAs, U3, U6other small stable RNAs

NucleoplasmIII

mRNAs, siRNA, U1, U2, U4,U5NucleoplasmII

28S, 18S, 5.8S rRNANucleolusI

ProductsLocationPolymerase

Mitochondria and plastids have "prokaryotic" RNA polymerases

E. coli promoter consensus sequences

RNA polymerase in start position

Initiation of transcription in E. coli

The ternary elongation complex

Eukaryotic transcription

Structure and organization of a eukaryotic gene

important sequence motifsin promotors

Transcription initiation in eukaryotesrequires core transcription factors

Interaction with enhancers is required for efficient transcriptioninitiation in eukaryotes

Enhancer and Insulator Sequences

RNA processing in eukaryotes

The basic transcription mechanism in eukaryotoes is similar to prokaryotes

Guanyltransferase adds 7-methylguanosin to 5' end

An endonuclease cuts approximately 20 nucleotides downstream of the polyA addition signal.

PolyA polymerase synthezises the poly(A) tail (about 200bp) to yieldthe complete primary mRNA (pre-mRNA).

Eukaryotic genes contain exons and introns

The ovalbumin gene is 7.7 kb long and encodes a mRNA of 1.9 kb.

The ovalbumin gene is 7.7 kb long and encodes a mRNAof 1.9 kb.

Splice consensus sequences

Conserved sequences in introns

Intron splicing in nuclear pre-mRNA occurs within a largeribonucleoprotein complex, the splicosome.

Splicing mechanism

Splicing is initiated by thenucleophilic attack of the 2'hydroxyl group of the branch siteadenosine on the phosphodiesterbond at the 5' splice site. Therebythe intron forms a lariat andreleases the 5' exon. Splicing iscompleted with a secondnucleophilic attack by the 3'-hydroxyl group of the 5' exon.

RNA splicing

Group I introns can self-splice (ribozymes);in mitochondria of yeast, rRNA genes of Tetrahymena, and plant organells

Group II introns and splicocomal introns share the same splicingmechanism;

in fungal mitochondria (self splicing), and in plant organells(not self splicing)

Three types of RNA splicing

An extensive network of coupling among gene expression machinesTom Maniatis, Robin Reed, Nature 416, 499 - 506 (2002)

The black arrows indicate physical and/or functional coupling

Gene expression factory model for coupling steps in gene expression.

PIC, preinitiation complexTF, transcription factorsCTD, carboxy-terminal domainCAP, capping factorSF, splicing factorpA, polyadenylation factorP, posphorylated CTD

CTD consists of hepta repeatsthat are phosphosylated duringinitiation (serin 5). This isrequired for binding the cappingapparatus. During elongation,serin 5 is dephosphorylated andserin 2 is phosphorylated, this isrequired for binding the splicingand poly(A) machineries.Genetic analysis: Deletion ofCTD decreases mRNAprocessing.

large RNAP subunit

PIC, preinitiation complexTF, transcription factorsCTD, carboxy-terminal domainCAP, capping factorSF, splicing factorpA, polyadenylation factorP, posphorylated CTD

CTD is dephosphorylatedduring termination

The mRNA is released as aribonucleoprotein

Model for couplingsplicing to mRNAexport and nonsense-mediated decay (NMD).

a) UAP recruits ALY andNMD

b) complex formation atexon/exon junction

c) NMD remains bound inthe cytoplasm

d) if NMD is not releasedby translation,degradation istriggered

mRNA degradation pathways

Reactions take place in the cytoplasm• shortening the polyA tail by 3' to 5' exonuclease• removal of CAP• degradation is completed by 5' to 3' exonuclease

Message

The mRNA of eukaryotes is processed by

• 5' capping• 3' polyadenylation• intron splicing

1. Three different RNApolymerases

2. mRNA is processed beforetransport to the cytoplasm

3. Genes contain often introns4. mRNAs are monocistronic

1. All RNAs are syntesized by asingle RNA polymerase

2. mRNA is translated duringtranscription

3. Genes are colinear with mRNA4. mRNA is often polycistronic

EukaryotesProkaryotes

Message

Is splicing an advantage for eukaryotes?

Exons often encode protein domains.

18 exons constitute the LDL receptor gene, containing six functional domains.

What is the evolutionary origin of splicing?

Intron early vs. intron late hypothesis.(Walter Gilbert)

AlternativAlternativ splicing splicing

products

e.g. adeno virus

Trans splicing

intron 1 intron 2

complementary intron sequences

trans splice products normal products

e.g. actin mRNA C. elegans

pre-mRNAs

Proteins

• Structure• Genetic Code• Translation

Protein structure

Protein structure

α-helix

Protein structure: antiparallel β sheet

Protein structure

Translation

The genetic code

tRNA

Structure of tRNA Allows Wobble in the Third PositionTransfer RNA recognizes the codons along mRNA and presents the correct amino acid for eachcodon. The first position of the anticodon on tRNA matches the third position of the codon.

FIGURE 2.16

Ribosome

Decoding centre; Peptidyl transfer centre

Mechanismus of peptide synthesisThe N3 of A2486 abstracts a proton from theNH2 group as the latter attacks the carbonylcarbon of the peptidyl-tRNA. (B) A protonatedN3 stabilizes the tetrahedral carbonintermediate by hydrogen bonding to theoxyanion. (C) The proton is transferred fromthe N3 to the peptidyl tRNA 3' OH as the newlyformed peptide deacylates.

Translation in Prokaryotes(A) Initiation of translation begins with the

association of the small ribosomesubunit with the Shine-Dalgarnosequence (S-D sequence) on themRNA. Next, the initiator tRNA thatreads AUG is charged with fMet. Thecharged initiator tRNA associates withthe small ribosome subunit and findsthe start codon. Assembly is helped byinitiation factors (IF1, IF2, and IF3)—notshown.

(B) During elongation peptide bonds areformed between the amino acids at theA-site and the P-site. The movement ofthe ribosome along the mRNA andaddition of a new tRNA to the A-site arecontrolled by elongation factors (alsonot shown). The E-site binds exitingtRNA.

(C) Termination requires release factors.The various components dissociate.The completed protein folds into itsproper three-dimensional shape.

Translation in Eukaryotes(A) Assembly of the small subunit plus

initiator Met-tRNA involves the bindingof factors eIF3 and eIF2.

(B) The cap binding protein of eIF4attaches to the mRNA before it joinsthe small subunit.

(C) The mRNA binds to the small subunitvia cap binding protein and the 40Sinitiation complex is assembled.

(D) Assembly of the large subunitrequires factor eIF5. After assembly,eIF2 and eIF3 depart.

Two more amino acids

selenocysteine pyrrolysine

COO-

+H3N C H

CH2

SeH

These are also called non-canonical amino acids

The genetic code for selenocysteine and pyrrolysine

selenocysteine insertion element (SECIS); pyrrolysine insertion element (PYLIS).

Codons that function usually as stop codons are used (in the context of aspecial structure of the mRNA) to encode these amino acids.

Nature (2004), 431, 257

Redefining the stop codon in mammalianmessenger RNAs. Ribosomes move alongmRNA, deciphering the nucleotide sequenceand making a protein according to theencoded amino-acid sequence. Thenucleotide sequence UGA normally specifiesthat the ribosome should stop translation.But sometimes this stop codon can beredefined, so that the twenty-first aminoacid — selenocysteine — is incorporatedinstead. This model shows how this mightbe done. a, A 'stem loop' structure in thedownstream, untranslated part of the mRNAbinds to a protein called SBP2. SBP2 in turnbinds to the eEFsec protein, which itself hasrecruited the transfer RNA carryingselenocysteine. b, The selenocysteine-bound tRNA is then delivered to the waitingUGA, for incorporation into the growingamino-acid string that constitutes the newlycreated protein.

When stop means go. There are two ways in which the stop codon UAGcould be redefined to specify the 22nd amino acid, pyrrolysine. In the first(top), special signals in mRNAs tag a subset of stop codons that are to havetheir meaning redefined. In the second (bottom), a codon is redefinedregardless of the mRNA involved.

A short consideration of genetic nomenclature

• gene• allele• wild type• wild type alleles• mutation• mutant alleles

Types of mutation

loss of function mutation

gain of function mutation

Types of mutation

• mutant site• leaky mutation• null mutation• silent mutation

Position of mutant sites and functional consequences

protein

active site

mutant site

promoter

neutral

null

leakynull (or leaky, or neutral)

gain of function