how cells read the genome: from dna to protein

How Cells Read the How Cells Read the Genome: From DNA to Genome: From DNA to

ProteinProtein

Chapter 6Chapter 6

How Cells Read the GenomeHow Cells Read the Genome

► TranscriptionTranscription► Translation Translation ► Folding of ProteinsFolding of Proteins► Evolution of the “Central Dogma”Evolution of the “Central Dogma”

How Cells Read the GenomeHow Cells Read the Genome

The Genomes of Multicellular Organisms: a State of Disarray!The Genomes of Multicellular Organisms: a State of Disarray!► IntronsIntrons► Irregularities in Gene DensityIrregularities in Gene Density► Little organization relating to gene function Little organization relating to gene function ► Adjacent genes often show no relatednessAdjacent genes often show no relatedness

How the Cells Read the GenomeHow the Cells Read the Genome

The “Central Dogma”The “Central Dogma”DNA RNA ProteinDNA RNA Protein

Variations in the Central Variations in the Central DogmaDogma

RNA SplicingRNA Splicing

RNA as the final gene productRNA as the final gene product

TranscriptionTranscription

RNA vs DNARNA vs DNA► Ribose as opposed to deoxribose sugarRibose as opposed to deoxribose sugar► Single strandedSingle stranded► Uracil in place of ThymineUracil in place of Thymine► RNA molecules w/ structural and catalytic propertiesRNA molecules w/ structural and catalytic properties


General Features of Transcription General Features of Transcription ► Produces RNA using one strand of DNA as template Produces RNA using one strand of DNA as template

moleculemolecule► Only small portion of DNA is transcribedOnly small portion of DNA is transcribed► Begins w/unwinding of sm portion of DNA exposing basesBegins w/unwinding of sm portion of DNA exposing bases► Transcript elongated by complementary base pairing by Transcript elongated by complementary base pairing by

RNA polymeraseRNA polymerase► RNA transcript shorter than DNA RNA transcript shorter than DNA


RNA PolymeraseRNA Polymerase► Transcribes DNA by catalyzing the formation phosphodiester bond Transcribes DNA by catalyzing the formation phosphodiester bond

btwn nucleotides btwn nucleotides ► Moves along DNA unwinding DNA just ahead of active siteMoves along DNA unwinding DNA just ahead of active site► Extends chain in the 5’ to 3’ directionExtends chain in the 5’ to 3’ direction► Hydrolysis of high-energy bonds provides energyHydrolysis of high-energy bonds provides energy► Many copies of RNA from same gene in small amount of timeMany copies of RNA from same gene in small amount of time


RNA Polymerase vs DNA PolymerseRNA Polymerase vs DNA Polymerse► RNA Polymerase catalyzes addition of ribonucleotidesRNA Polymerase catalyzes addition of ribonucleotides► Can begin RNA synthesis without primerCan begin RNA synthesis without primer► Not as accurate as DNA Polymerse; RNA Polymerase 1 error/10Not as accurate as DNA Polymerse; RNA Polymerase 1 error/1044

nucleotides vs 1 error/10nucleotides vs 1 error/1077 nucleotides nucleotides► Both have proof reading capabilityBoth have proof reading capability


Different Types of RNADifferent Types of RNA► mRNA; RNA copied from genes that ultimately direct synthesis of mRNA; RNA copied from genes that ultimately direct synthesis of

proteins (3-5% total RNA)proteins (3-5% total RNA)► Final product of minority of genes is RNA itself: tRNA, rRNA, snRNA Final product of minority of genes is RNA itself: tRNA, rRNA, snRNA

(majority of total RNA)(majority of total RNA)► Tens of thousands of different mRNA transcripts; 10-15 copies of ea Tens of thousands of different mRNA transcripts; 10-15 copies of ea

species/cellspecies/cell

Transcription in ProcaryotesTranscription in Procaryotes

Start and Stop Signals embedded in DNA Start and Stop Signals embedded in DNA ► Promoter= DNA sequence RNA Polymerase binds to initiate RNA Promoter= DNA sequence RNA Polymerase binds to initiate RNA

synthesissynthesis► RNA Polymerase’s sigma subunit (bact) binds to promoter & opens helixRNA Polymerase’s sigma subunit (bact) binds to promoter & opens helix► One of exposed strands serves as templateOne of exposed strands serves as template► After synthsis of ~10 bases sigma subunit of RNA Polymerase After synthsis of ~10 bases sigma subunit of RNA Polymerase

disassociatesdisassociates► RNA Polymerase undergoes structural changes moving forward rapidly RNA Polymerase undergoes structural changes moving forward rapidly

synthesizing RNA at ~50 bp/secsynthesizing RNA at ~50 bp/sec► RNA Polymerase continues until termination signal of string of A-T RNA Polymerase continues until termination signal of string of A-T

preceded by “hairpin loop”preceded by “hairpin loop”► Termination signal causes RNA Pol to disassociates from RNA releasing Termination signal causes RNA Pol to disassociates from RNA releasing

DNA DNA ► Free RNA Polym complexes once again w/ sigma subunitFree RNA Polym complexes once again w/ sigma subunit

Transcription in ProcaryotesTranscription in Procaryotes


Transcription Promoter and Terminator SignalsTranscription Promoter and Terminator Signals► Heterogeneous but contain related consensus sequence recognized Heterogeneous but contain related consensus sequence recognized

by sigma subunit of RNA Polby sigma subunit of RNA Pol► Precise promoter sequence governs affinity for RNA Pol = Precise promoter sequence governs affinity for RNA Pol =

“strength”“strength”► Can be predicted by algorithms but need to be independently Can be predicted by algorithms but need to be independently

verifiedverified► Promoters are assymetric; RNA Pol can bind in one orientation and Promoters are assymetric; RNA Pol can bind in one orientation and

extend in 5’ to 3’ direction onlyextend in 5’ to 3’ direction only► Terminators more heterogeneous than promoters; ability of RNA to Terminators more heterogeneous than promoters; ability of RNA to

fold into “hairpin loop” is most important featurefold into “hairpin loop” is most important feature

Transcription in EucaryotesTranscription in Eucaryotes

3 Types of RNA Polymerases of similar in structure in 3 Types of RNA Polymerases of similar in structure in EucryotesEucryotes

► RNA Pol I- transcribes tRNA, rRNA, smRNAsRNA Pol I- transcribes tRNA, rRNA, smRNAs► RNA Pol II- transcribes genes encoding proteinsRNA Pol II- transcribes genes encoding proteins► RNA Pol III- transcribes tRNA, rRNA, smRNAsRNA Pol III- transcribes tRNA, rRNA, smRNAs


Transcription in Eukaryotes vs ProcaryotesTranscription in Eukaryotes vs Procaryotes► Nucleosomes and higher order chromatin packagingNucleosomes and higher order chromatin packaging► RNA Pol requires General Transcription FactorsRNA Pol requires General Transcription Factors


General Transcription Factors (Eukaryotes)General Transcription Factors (Eukaryotes)► Assemble at promoters of all RNA Pol II transcriptsAssemble at promoters of all RNA Pol II transcripts► Facilitate binding of RNA Pol IIFacilitate binding of RNA Pol II► Aid in opening DNA strands for transcription to beginAid in opening DNA strands for transcription to begin► Release RNA Pol from promoter into elongation mode Release RNA Pol from promoter into elongation mode

once transcription has begunonce transcription has begun


5 different General Transcription Factors


Other Proteins Required by RNA Pol in EucaryotesOther Proteins Required by RNA Pol in Eucaryotes1.1. Transcriptional activators- bind to specific sequences and facilitate Transcriptional activators- bind to specific sequences and facilitate

binding of GTFs and RNA Polbinding of GTFs and RNA Pol2.2. Mediators- enable activators to interact w/ GTFs and RNA PolMediators- enable activators to interact w/ GTFs and RNA Pol3.3. Chromatin Remodeling Complexes- allow greater accessibility to DNAChromatin Remodeling Complexes- allow greater accessibility to DNA4.4. Many proteins required to initiate transcription, > 100 subunitsMany proteins required to initiate transcription, > 100 subunits


ElongationElongation► Elongation factors= ensures that RNA Pol does not disassociate Elongation factors= ensures that RNA Pol does not disassociate

before end of gene; assoc. w/ RNA Pol shortly after initiation of before end of gene; assoc. w/ RNA Pol shortly after initiation of transcriptiontranscription

► DNA topoisomerases removes superhelical tensionDNA topoisomerases removes superhelical tension► DNA gyrases uses ATP to pump supercoils into DNADNA gyrases uses ATP to pump supercoils into DNA► Elongation tightly coupled to RNA processing Elongation tightly coupled to RNA processing


RNA ProcessingRNA Processing1.1. Eucaryotic mRNA capped at 5’ end Eucaryotic mRNA capped at 5’ end 2.2. polyadenylated at 3’ endpolyadenylated at 3’ end3.3. Introns removedIntrons removed

Phosphorylation of RNA tail CTDPhosphorylation of RNA tail CTD► consists of domain of repeated 52 times of 7 aa consists of domain of repeated 52 times of 7 aa

containing 2 serines that are phosphorylatedcontaining 2 serines that are phosphorylated► phosphorylation of tail promotes disassoc of RNAphosphorylation of tail promotes disassoc of RNA Pol from proteins presents at start of transcriptionPol from proteins presents at start of transcription► allows new set of proteins that function in allows new set of proteins that function in

elongation and pre-mRNA processeing, to assocelongation and pre-mRNA processeing, to assoc


Capping of Pre-mRNAsCapping of Pre-mRNAs► Capping of 5’ end w/ modified guanine nucleotide occurs after ~25 bases Capping of 5’ end w/ modified guanine nucleotide occurs after ~25 bases

synthesizedsynthesized► 3 enzymes involved in capping process3 enzymes involved in capping process

1. phosphatase removes one P’ from 5’ end of RNA1. phosphatase removes one P’ from 5’ end of RNA 2.2. guanyl transferase adds GMP to 5’ endguanyl transferase adds GMP to 5’ end 3.3. methyl transferase adds methyl grp to guanosinemethyl transferase adds methyl grp to guanosine

► Capping enzymes bind to phosphorylated tail of RNA PolCapping enzymes bind to phosphorylated tail of RNA Pol► Cap binds CBC (cap binding complex) that facilitates RNA processing and Cap binds CBC (cap binding complex) that facilitates RNA processing and

exportexport


General Features of RNA SplicingGeneral Features of RNA Splicing► Involves two transesterification reactions Involves two transesterification reactions

to join exons and remove intron in form to join exons and remove intron in form of lariatof lariat

► 5 additional RNAs, > 50 proteins, and 5 additional RNAs, > 50 proteins, and lots of ATP requiredlots of ATP required

► Complexity ensures accuracyComplexity ensures accuracy► Alternative splicing occurs in 60% of Alternative splicing occurs in 60% of

human geneshuman genes► Increase coding potential and facilitates Increase coding potential and facilitates

evol of new protein sequencesevol of new protein sequences


Sequences Mark Where Splicing OccursSequences Mark Where Splicing Occurs► Intron size varies from 10-100,000 Intron size varies from 10-100,000

nucleotidesnucleotides► 3 conserved nucleotide sequences3 conserved nucleotide sequences

5’ splice site5’ splice site

3’ splice site3’ splice site

branch points that forms base of excised branch points that forms base of excised lariatlariat


Spliceosome Mediates Splicing of RNASpliceosome Mediates Splicing of RNA► performed primarily by 5 snRNA molec performed primarily by 5 snRNA molec

(U1, U2, U4, U5) forming spliceosome core (U1, U2, U4, U5) forming spliceosome core ► snRNA molec recognize intron-exon snRNA molec recognize intron-exon

borders and participate in splicing borders and participate in splicing chemistrychemistry

► Spliceosome complex of RNA and proteinSpliceosome complex of RNA and protein► More than 50 proteins invovledMore than 50 proteins invovled


Mechanism of RNA SlicingMechanism of RNA Slicing1. spliceosome recognizes splicing signals 1. spliceosome recognizes splicing signals

on pre-mRNA, brings ends of intron on pre-mRNA, brings ends of intron togethertogether

2. branch point site recognized by BBP 2. branch point site recognized by BBP and U2AFand U2AF

3.3. U2 snRNP displaces BBP base pairing w/ U2 snRNP displaces BBP base pairing w/ branch point consensus seqbranch point consensus seq

4.4. U1 snRNP base pairs w/ 5’ splice site U1 snRNP base pairs w/ 5’ splice site junctionjunction

5. U4/U6•U5 triple snRNP enters5. U4/U6•U5 triple snRNP enters6.6. RNA-RNA rearrangements disrupts RNA-RNA rearrangements disrupts

U4/U6 base pairing to enable U6 to U4/U6 base pairing to enable U6 to displace U1 at 5’ splice junction; U4 displace U1 at 5’ splice junction; U4 exitsexits

7. U2 and U6 snRNPs in spliceosome form 7. U2 and U6 snRNPs in spliceosome form 3d RNA structure bringing 5’ junction 3d RNA structure bringing 5’ junction into position near branch chain A for into position near branch chain A for first esterificationfirst esterification


Mechanism of RNA SlicingMechanism of RNA Slicing 8. 5’ and 3’ junctions brought 8. 5’ and 3’ junctions brought together together via U5 snRNP for second via U5 snRNP for second esterificationesterification9. snRNPs remain bound to lariat 9. snRNPs remain bound to lariat whilewhile splice product releasedsplice product released


Spliceosome and ATP HydrolysisSpliceosome and ATP Hydrolysis► Not required for splicing chemistryNot required for splicing chemistry► Needed for assembly and rearrangements Needed for assembly and rearrangements ► RNA helicase requiring ATP needed to break RNA-RNA RNA helicase requiring ATP needed to break RNA-RNA

interactionsinteractions► All steps except assoc of BBP w/ branch chain A, and U2 w/ 5’ All steps except assoc of BBP w/ branch chain A, and U2 w/ 5’

splice site require ATP and other proteinssplice site require ATP and other proteins► Removal of snRNP from lariat requires RNA-RNA interactions Removal of snRNP from lariat requires RNA-RNA interactions

that are dependent on ATP hydrolysisthat are dependent on ATP hydrolysis

TranscriptionTranscriptionAssembly of SpliceosomeAssembly of Spliceosome► Occurs as pre-mRNA emerges from transcribing RNA PolOccurs as pre-mRNA emerges from transcribing RNA Pol► Components of the spliceosome are carried on RNA Pol tail and transferred Components of the spliceosome are carried on RNA Pol tail and transferred

to nascent pre-mRNAto nascent pre-mRNA► Exon size tend to be uniform ~150 bpExon size tend to be uniform ~150 bp► Spliceosome assembly occurs co-transcriptionally, splicing sometimes occurs Spliceosome assembly occurs co-transcriptionally, splicing sometimes occurs

post-transcriptionallypost-transcriptionally► Spliceosome proteins SR (rich in Ser and Arg) assemble on exon and mark Spliceosome proteins SR (rich in Ser and Arg) assemble on exon and mark

off 3’ and 5’ site starting at 5’ end of mRNA, assembly occurs in conjunction off 3’ and 5’ site starting at 5’ end of mRNA, assembly occurs in conjunction w/ U1 snRNA and U2AFw/ U1 snRNA and U2AF


Plasticity of RNA SplicingPlasticity of RNA Splicing► Splicing mech selected for flexibilitySplicing mech selected for flexibility► Flexibility enables cell to regulate pattern of RNA splicingFlexibility enables cell to regulate pattern of RNA splicing► Alternative splicing when diff proteins can be made from Alternative splicing when diff proteins can be made from

same genesame gene► Splicing patters regulated so diff forms of protein produced at Splicing patters regulated so diff forms of protein produced at

diff times and in diff tissuesdiff times and in diff tissues


Self-Splicing IntronsSelf-Splicing Introns► Group I Intron= reactive G nucleotide attacks the initial Group I Intron= reactive G nucleotide attacks the initial

phosphdiester bound cleaved during the spicing rxnphosphdiester bound cleaved during the spicing rxn► Group II Intron= reactive A in intron seq is attaching grp, and Group II Intron= reactive A in intron seq is attaching grp, and

lariat intermediate generatedlariat intermediate generated► Sequence of self=splicing introns is critical; RNA folds in Sequence of self=splicing introns is critical; RNA folds in

specific 3d conformation that brings 5’ and 3’ junctions specific 3d conformation that brings 5’ and 3’ junctions together and provides precisiely positioned reactive grps to together and provides precisiely positioned reactive grps to perform chemistryperform chemistry

► Pre-mRNA splicing mech evolved from Grp II SplicingPre-mRNA splicing mech evolved from Grp II Splicing

spliceosomal snRNPs took over structural and chemical roles spliceosomal snRNPs took over structural and chemical roles of Grp II Introns so sequence constraints no longer neededof Grp II Introns so sequence constraints no longer needed


Group I Introns Group II Introns


Processing of 3’ End of pre-mRNAProcessing of 3’ End of pre-mRNA► Termination signs are transcribed into RNA and recognized proteins Termination signs are transcribed into RNA and recognized proteins

as RNA Pol transcribes thru themas RNA Pol transcribes thru them► CstF (cleavage stimulating factor F) and CPSF (cleavage and CstF (cleavage stimulating factor F) and CPSF (cleavage and

processing specificity factor) proteins assoc w/ RNA Pol tail processing specificity factor) proteins assoc w/ RNA Pol tail transferred to RNA as it emergestransferred to RNA as it emerges


Processing the 3’ End of the Pre-mRNAProcessing the 3’ End of the Pre-mRNA► Additional proteins assemble w/ CstF and Additional proteins assemble w/ CstF and

CPSF to perform processing:CPSF to perform processing:

1. RNA is cleaved1. RNA is cleaved

2. Poly-A-Polymerase adds ~200 A’s to 3’ 2. Poly-A-Polymerase adds ~200 A’s to 3’ end ofend of

cleaved productcleaved product

3. RNA Pol II continues to transcribe after 3. RNA Pol II continues to transcribe after pre-pre-

mRNA has been mRNA has been

cleaved; several 100 bases before falls offcleaved; several 100 bases before falls off

template and transcription terminatestemplate and transcription terminates

4. RNA downstream of cleavage is degraded4. RNA downstream of cleavage is degraded

TranscriptionTranscriptionSelective Export of Mature mRNAs from NucleusSelective Export of Mature mRNAs from NucleusHow does cell distinguish btwn rate mature mRNA and debris from mRNAHow does cell distinguish btwn rate mature mRNA and debris from mRNAprocessing?processing?► Export highly selected and coupled to correct mRNA processingExport highly selected and coupled to correct mRNA processing► mRNA exported only if appr set of proteins are bound including: 1) cap mRNA exported only if appr set of proteins are bound including: 1) cap

binding complex 2) snRNP proteins absent 3) proteins that mark complete binding complex 2) snRNP proteins absent 3) proteins that mark complete splicingsplicing

Selective Export of Mature mRNAs from NucleusSelective Export of Mature mRNAs from Nucleus


hnRNPs= heterogeneous nuclear ribonuclear proteins, most hnRNPs= heterogeneous nuclear ribonuclear proteins, most abundantabundant

proteins that assemble on pre-mRNA as it emerges from RNA Polproteins that assemble on pre-mRNA as it emerges from RNA Pol► Some hnRNPs remove hairpin helices from RNA so that splicing Some hnRNPs remove hairpin helices from RNA so that splicing

and other signals on RNA can be readand other signals on RNA can be read► hnRNPs excluded from exons, remain on excised introns hnRNPs excluded from exons, remain on excised introns

marking them for nuclear retention and/or destructionmarking them for nuclear retention and/or destruction► Some reamin bound to fully processed mRNA and accompany Some reamin bound to fully processed mRNA and accompany

them to cytoplasmthem to cytoplasm


Most of the RNA in the Cell Performs a Catalytic or Structural Most of the RNA in the Cell Performs a Catalytic or Structural FunctionFunction

► ~80% of total RNA is rRNA~80% of total RNA is rRNA► 3-5% of total RNA mRNA3-5% of total RNA mRNA► rRNA transcribed by RNA Pol I (which has no C-terminal tail)rRNA transcribed by RNA Pol I (which has no C-terminal tail)► rRNA is neither capped or polyadenylatedrRNA is neither capped or polyadenylated► RNA components of ribosome are final gene products; RNA components of ribosome are final gene products;

growing cell syn ~10 million of ea type of rRNA ea cell growing cell syn ~10 million of ea type of rRNA ea cell generationgeneration


rRNA GenesrRNA Genes► Mammalian cells contain 10 million ribosomesMammalian cells contain 10 million ribosomes► Multi-copy genes Multi-copy genes

E. coli has 7 copies of its rRNAE. coli has 7 copies of its rRNAHumans ~200 copies on 5 chromosomesHumans ~200 copies on 5 chromosomesXenopus ~600 copies single cluster on 1 chromosomeXenopus ~600 copies single cluster on 1 chromosome

► 4 types of eucaryotic rRNAs ea present in one copy/ribosome4 types of eucaryotic rRNAs ea present in one copy/ribosome18S, 5.8S and 28S encoded by single lg precursor RNA-chemically modified18S, 5.8S and 28S encoded by single lg precursor RNA-chemically modified5S rRNA syn from separate cluster by Pol III- not chemically modified5S rRNA syn from separate cluster by Pol III- not chemically modified


Chemical Modification of Lg rRNA PrecursorChemical Modification of Lg rRNA Precursor► 100 methylations of 2’-OH100 methylations of 2’-OH► 100 isomerizations of uridines100 isomerizations of uridines► Function of chem. modification unknwn but Function of chem. modification unknwn but

may facilitate folding or assemblymay facilitate folding or assembly► snoRNAs= small nucleolar RNAs guide in snoRNAs= small nucleolar RNAs guide in

chemical modification and cleavage of chemical modification and cleavage of precursor rRNAprecursor rRNA

► snoRNAs encoded in introns, esp those of snoRNAs encoded in introns, esp those of ribosomal proteins, function in nucleolusribosomal proteins, function in nucleolus


Nucleolus as a Ribosome Producing FactoryNucleolus as a Ribosome Producing Factory► site for processing rRNAs and assembly into site for processing rRNAs and assembly into

ribosomesribosomes► lg aggregate of macromolecules including: lg aggregate of macromolecules including:

rRNA genes, precursor rRNAs, mature rRNAs, rRNA genes, precursor rRNAs, mature rRNAs, rRNA processing enzymes, snoRNPs, rRNA processing enzymes, snoRNPs, ribosomal protein subunits, and some ribosomal protein subunits, and some partially assembled ribosomespartially assembled ribosomes

► Size varies and reflects number of ribosomes Size varies and reflects number of ribosomes cell is making; may occupy 25% of nuclear cell is making; may occupy 25% of nuclear volvol

► Also site where other RNAs produced and Also site where other RNAs produced and other RNA-protein complexes assembled other RNA-protein complexes assembled (tRNAs, snoRNAs, U6 snRNP, telomerase)(tRNAs, snoRNAs, U6 snRNP, telomerase)

TranslationTranslation

From RNA to ProteinFrom RNA to Protein► Genetic code dictates how mRNA translated into aa sequence of Genetic code dictates how mRNA translated into aa sequence of

proteinprotein► Nucleotides read consecutively in grps of 3 (condons)Nucleotides read consecutively in grps of 3 (condons)► Degenerate genetic code; 64 codons specify 20 aaDegenerate genetic code; 64 codons specify 20 aa► 6 possible reading frames6 possible reading frames


tRNA as an Adaptor MoleculetRNA as an Adaptor Molecule► Recognizes and binds to codon and appr. aaRecognizes and binds to codon and appr. aa► ~80 nucleotdies~80 nucleotdies► Folds into clover leaf and then into L-shaped structureFolds into clover leaf and then into L-shaped structure► Two regions of unpaired nucelotdies at ends of L shaped molecule essential to Two regions of unpaired nucelotdies at ends of L shaped molecule essential to

functionfunction

1. anticodon= set of 3 consecutive nucleotides pairs w/complementary 1. anticodon= set of 3 consecutive nucleotides pairs w/complementary codon codon on mRNA on mRNA

2. short ss region near 3’end where aa attaches2. short ss region near 3’end where aa attaches


Redundancy/Degeneracy of Genetic CodeRedundancy/Degeneracy of Genetic Code► More than one tRNA for many of the aaMore than one tRNA for many of the aa► Some tRNAs base pair w/ more than one aa; Wobble PositionSome tRNAs base pair w/ more than one aa; Wobble Position


tRNAstRNAs► Number and kinds of tRNAs varies across Number and kinds of tRNAs varies across

species ie: humans have 497 tRNA genes w/ species ie: humans have 497 tRNA genes w/ only 48 diff anticodons representedonly 48 diff anticodons represented

► Transcribed by RNA Pol IIITranscribed by RNA Pol III► Some transcripts are spliced via cut-and-paste Some transcripts are spliced via cut-and-paste

mechanism catalyzed by proteins (no lariat) mechanism catalyzed by proteins (no lariat) occurs when tRNA is properly folded in occurs when tRNA is properly folded in cloverleaf confcloverleaf conf

► Extensive modifications > 50 different kindsExtensive modifications > 50 different kinds► 1 modification/10 nucleotides1 modification/10 nucleotides► Modifications essential to: Modifications essential to:

1) accuracy of tRNA attaching to correct aa 1) accuracy of tRNA attaching to correct aa

2) recognition of mRNA codon by tRNA 2) recognition of mRNA codon by tRNA anticodonanticodon


Aminoacyl-tRNA SynthetasesAminoacyl-tRNA Synthetases► Couple aa to appr set of tRNAsCouple aa to appr set of tRNAs► Aa specificAa specific► Enzymatic reaction attaches aa to 3’ end of tRNA requires ATPEnzymatic reaction attaches aa to 3’ end of tRNA requires ATP► High energy bond produced btwn aa and tRNA later used to covalently High energy bond produced btwn aa and tRNA later used to covalently

link aa to growing polypeptide chainlink aa to growing polypeptide chain


Editing by tRNA synthetases►Selects correct aa in two step process:

1. correct aa highest affinity for active site

2. aa forced into second pocket whose

dimensions exclude correct aa

►Those that enter second editing site

are hydrolyzed from AMP- hydrolytic editing

►Raises overall accuracy of tRNA charging to

1 mistake/40,000 couplings

►Synthetase must also recognize correct tRNAs


Ribosome Structure: rRNA molecules + 50 different Ribosome Structure: rRNA molecules + 50 different proteinsproteins


Ribosome Structure and FunctionRibosome Structure and Function► Large and Small Subunits; rRNA sequence highly conservedLarge and Small Subunits; rRNA sequence highly conserved► 66% RNA; 33% protein66% RNA; 33% protein► rRNA responsible for:rRNA responsible for:

structurestructurepositioning tRNAs on mRNApositioning tRNAs on mRNAcatalytic activity in forming peptide bondcatalytic activity in forming peptide bond


mRNA Decoded on RibosomesmRNA Decoded on Ribosomes► Ribosomal subunits assemble on mRNA near 5’ endRibosomal subunits assemble on mRNA near 5’ end► Ribosome translates mRNA in aa sequence using tRNAs as adaptors Ribosome translates mRNA in aa sequence using tRNAs as adaptors

to add aa in correct sequence to end of growing polypeptide chainto add aa in correct sequence to end of growing polypeptide chain► aa linked by peptide bond formation aa linked by peptide bond formation ► 2 aa added/sec eucaryotic cell; 20 aa/sec added by bact2 aa added/sec eucaryotic cell; 20 aa/sec added by bact► 4 impt ribosomal binding sites4 impt ribosomal binding sites


Major Steps in TranslationMajor Steps in Translation

1.1. Ribosomal Assembly and InitaitionRibosomal Assembly and Initaition

2.2. ElongationElongation

3.3. Termination and Release of Nascent PolypeptideTermination and Release of Nascent Polypeptide


Ribosomal AssemblyRibosomal Assembly1.1. Initiation tRNA-Met & eIFs bind sm rRNA Initiation tRNA-Met & eIFs bind sm rRNA

subunitsubunit

2.2. Sm rRNA subunit binds to 5’ end of mRNA Sm rRNA subunit binds to 5’ end of mRNA recognizing CAP and 2 eIFs (eucaryotes)recognizing CAP and 2 eIFs (eucaryotes)

3.3. Sm rRNA scans for AUG startSm rRNA scans for AUG start

4.4. eIFs dissociate and lg rRNA bindseIFs dissociate and lg rRNA binds

5.5. Initiator tRNA-met now in P-site leaving A-Initiator tRNA-met now in P-site leaving A-site vacant for incoming aminoacyl rRNA to site vacant for incoming aminoacyl rRNA to start protein synthesisstart protein synthesis


Procaryotes have Shine Delgarno SequenceProcaryotes have Shine Delgarno Sequence► Specific sequence few nucleotides upstream of AUG startSpecific sequence few nucleotides upstream of AUG start► AGGAGGUAGGAGGU► Binding site positions sm rRNA subunit at AUG startBinding site positions sm rRNA subunit at AUG start► Bacterial mRNAs can be poly-cistronic; eucaryotes monocistronicBacterial mRNAs can be poly-cistronic; eucaryotes monocistronic


ElongationElongation1.1. tRNA carrying next aa in chain tRNA carrying next aa in chain

binds to ribo A-site base pairing binds to ribo A-site base pairing w/ mRNA codonw/ mRNA codon

2.2. Peptide Bond Formation= Peptide Bond Formation= carboxyl end released from tRNA carboxyl end released from tRNA at P-site and joined to free amino at P-site and joined to free amino grp of aa linked to tRNA at A site grp of aa linked to tRNA at A site forming new peptide bondforming new peptide bond

3.3. Translocation= conformational Translocation= conformational chgs move mRNA 3 nucleotides chgs move mRNA 3 nucleotides along ribo resetting ribo for next along ribo resetting ribo for next acyl tRNAacyl tRNA


Two Impt Elongation Factors Drive TranslationTwo Impt Elongation Factors Drive Translation► EF-Tu Mechanism- resp. 99% accuracy EF-Tu Mechanism- resp. 99% accuracy

a. Chged tRNA enters ribo bound to GTP forma. Chged tRNA enters ribo bound to GTP form

b. Codon recognition triggers GTP hydrolysisb. Codon recognition triggers GTP hydrolysis

c. EF-Tu dissociates from ribo w/out tRNAc. EF-Tu dissociates from ribo w/out tRNA

d. Introduces 2 short delays btwn codon-d. Introduces 2 short delays btwn codon- anticodon pairing and chain elongation anticodon pairing and chain elongation

► EF-G binds in or near A site hydrolyzes GTP whose EF-G binds in or near A site hydrolyzes GTP whose energy accelerates movement of bound tRNAs in A/P energy accelerates movement of bound tRNAs in A/P and P/E hybrid statesand P/E hybrid states


TerminationTermination► Stop codons: UAA, UAG, UGAStop codons: UAA, UAG, UGA► Stop codons recognized by “release factor” Stop codons recognized by “release factor”

proteinsproteins► Release factors cause peptidyl transferase to Release factors cause peptidyl transferase to

catalyze addition of water to peptidyl tRNA catalyze addition of water to peptidyl tRNA ► Hydrolysis frees COOH end of polypeptide Hydrolysis frees COOH end of polypeptide

chain from attachment to tRNAchain from attachment to tRNA► Ribosomal dissassemblyRibosomal dissassembly


Proteins are Made on PolyribosomesProteins are Made on Polyribosomes► Syn of avg protein varies 20 sec to 2-3 minSyn of avg protein varies 20 sec to 2-3 min► mRNAs translated in form of polyribosomes w/ ribos spaced mRNAs translated in form of polyribosomes w/ ribos spaced >>

80 nucleotides80 nucleotides► Transcription and translation occur simultaneously in Transcription and translation occur simultaneously in

procaryotesprocaryotes


Quality Control of TranslationQuality Control of Translation► Accuracy ~1 mistake/10Accuracy ~1 mistake/1044 aa aa► Speed of translation 20 aa incorporated/sec in pro. vs 2 aa/sec Speed of translation 20 aa incorporated/sec in pro. vs 2 aa/sec

euk.euk.► Protein synthesis consumes more energy than any other Protein synthesis consumes more energy than any other

biosynthetic process; 4 ATP equivalents/ aabiosynthetic process; 4 ATP equivalents/ aa► Quality control mech to ensure mRNA is complete recognition Quality control mech to ensure mRNA is complete recognition

of Cap and poly A tail by initiation complexof Cap and poly A tail by initiation complex


Antibiotics

►Inhibitors of bacterial protein synthesis are effective antibiotics

►Specificity of antibiotics useful for molecular cell biology studies

Life and Death of ProteinsLife and Death of Proteins

Protein MaturationProtein Maturation► Protein folding begins while protein is being synthesizedProtein folding begins while protein is being synthesized► Maturation:Maturation:

unique 3d structureunique 3d structure

bind sm moleculesbind sm molecules

modificationsmodifications

assemble w/ other proteinsassemble w/ other proteins


Protein FoldingProtein Folding► Steps in protein folding: Steps in protein folding:

1. molten globule1. molten globule

2. slow phase2. slow phase► Most of folding complete by time Most of folding complete by time

released from ribosomereleased from ribosome► Molecular chaperones held guide Molecular chaperones held guide

folding of many proteinsfolding of many proteins


Molecular ChaperoninsMolecular Chaperonins► Two major families: hsp60 & Two major families: hsp60 &

hsp70hsp70► Affinity for exposed hydrophobic Affinity for exposed hydrophobic

patches patches ► Massage protein into folded Massage protein into folded

conformation via ATP hydrolysisconformation via ATP hydrolysis► Hsp70 acts early in life of protein; Hsp70 acts early in life of protein;

binds to stretch of 7 hydrophobic binds to stretch of 7 hydrophobic aa before protein leaves riboaa before protein leaves ribo

► Hsp60 acts later in proteins life, Hsp60 acts later in proteins life, forms barrel into which proteins forms barrel into which proteins fedfed


ProteosomeProteosome► Protein disposal apparatus dispersed throughout cytosoloProtein disposal apparatus dispersed throughout cytosolo► Hollow cylinder of proteases form stack of 4 heptameric ringsHollow cylinder of proteases form stack of 4 heptameric rings► Ends composed of protein complex of ~20 polypeptides, 6 of which Ends composed of protein complex of ~20 polypeptides, 6 of which

hydrolyze ATP to unfold proteins and move them into proteosomehydrolyze ATP to unfold proteins and move them into proteosome► Acts on proteins marked by ubiquitinActs on proteins marked by ubiquitin


Ubiquitin conjugating systemUbiquitin conjugating system► Multiubiquitin chain on target proteins recognized by receptors on Multiubiquitin chain on target proteins recognized by receptors on

proteosomeproteosome► Targeted proteins: misfolded, oxidized, or other abnormal aaTargeted proteins: misfolded, oxidized, or other abnormal aa► Linear series of ubiquitin conjugates linked via lysine on target proteinLinear series of ubiquitin conjugates linked via lysine on target protein


Regulated DestructionRegulated Destruction► Activation of compments in proteolytic pathway E1, E2, E3Activation of compments in proteolytic pathway E1, E2, E3► Degradation signals created in response to intracellular or Degradation signals created in response to intracellular or

extracellular signalsextracellular signals► N-terminal ruleN-terminal rule

12 destabilizing aa including Arg, Lys, His, Leu, Asp Try, Tyr, Asp, 12 destabilizing aa including Arg, Lys, His, Leu, Asp Try, Tyr, Asp, Glu, Asn, GlnGlu, Asn, Gln

how cells read the genome: from dna to protein

Documents

dna sequence rna polymerase

synthesizing rna

dna polymerse rna polymerase

assymetric rna pol

dna unwinding dna

dna promoter

energymany copies of

promoters ability of