Dr. SharmaJan,09 Overview: The Flow of Genetic Information The information content of DNA Is in the form of specific sequences of nucleotides along the.

Dr. Sharma Jan,09

Overview:

The Flow of Genetic Information The information content of DNA

Is in the form of specific sequences of nucleotides along the DNA strands

Dr. Sharma Jan,09

The DNA inherited by an organismLeads to specific traits by dictating the synthesis of proteins

The process by which DNA directs protein synthesis, gene expression

Includes two stages, called transcription and translation

Dr. Sharma Jan,09

The ribosomeThe ribosomeIs part of the cellular machinery for Is part of the cellular machinery for translation, polypeptide synthesistranslation, polypeptide synthesis

Dr. Sharma Jan,09

Genes specify proteins via transcription and translation

Dr. Sharma Jan,09

TranscriptionTranscriptionIs the synthesis of RNA under the direction Is the synthesis of RNA under the direction of DNAof DNAProduces messenger RNA (mRNA)Produces messenger RNA (mRNA)

TranslationTranslationIs the actual synthesis of a polypeptide, Is the actual synthesis of a polypeptide, which occurs under the direction of mRNAwhich occurs under the direction of mRNAOccurs on ribosomesOccurs on ribosomes

Dr. Sharma Jan,09

prokaryotesprokaryotesTranscription and translation occur togetherTranscription and translation occur together

Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing.

(a)

TRANSLATION

TRANSCRIPTIONDNA

mRNA

Ribosome

Polypeptide

Dr. Sharma Jan,09

EukaryotesEukaryotesRNA transcripts are modified before RNA transcripts are modified before becoming true mRNAbecoming true mRNA

Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA.

(b)

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

mRNA

DNA

Pre-mRNA

Polypeptide

Ribosome

Nuclearenvelope

Dr. Sharma Jan,09

Cells are governed by a cellular chain of command

DNA RNA protein

Dr. Sharma Jan,09

Genetic informationIs encoded as a sequence of non overlapping base triplets, or codons

Dr. Sharma Jan,09

The gene determines the The gene determines the sequence of bases along the sequence of bases along the length of an mRNA moleculelength of an mRNA molecule

Figure 17.4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

TRANSCRIPTION

mRNA

Protein

TRANSLATION

Amino acid

A C C A A A C C G A G T

U G G U U U G G C U C A

Trp Phe Gly Ser

Codon

3 5

35

Dr. Sharma Jan,09

A codon in messenger RNAA codon in messenger RNAIs either translated into an amino acid or Is either translated into an amino acid or serves as a translational stop signalserves as a translational stop signal

Second mRNA baseU C A G

U

C

A

G

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

Met orstart

Phe

Leu

Leu

lle

Val

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

Ser

Pro

Thr

Ala

UAUUAC

UGUUGC

Tyr Cys

CAUCACCAACAG

CGUCGCCGACGG

AAUAACAAAAAG

AGUAGCAGAAGG

GAUGACGAAGAG

GGUGGCGGAGGG

UGGUAAUAG Stop

Stop UGA StopTrp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

GUCAG

UCAG

UCAG

Fir

st m

RN

A b

ase

(5

end

)

Th

ird

mR

NA

bas

e (3

en

d)

Glu

Dr. Sharma Jan,09

Codons must be read in the correct reading frame

For the specified polypeptide to be produced

Dr. Sharma Jan,09

The genetic code The genetic code

is nearly universalis nearly universal

Non-overlappNon-overlappDegenerate-Degenerate-- more than one codon specifies/ codes for a particular amino acid

--Shared by organisms from the Shared by organisms from the simplest bacteria to the most simplest bacteria to the most complex animalcomplex animal

Dr. Sharma Jan,09

Molecular Components of Transcription

RNA synthesisIs catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotidesFollows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

Dr. Sharma Jan,09

Synhesis of an RNA Synhesis of an RNA TranscriptTranscript

InitiationElongationTermination

PromoterTranscription unit

RNA polymerase

Start point

53

35

35

53

53

35

53

35

5

5

Rewound

DNA

RNA

transcript

3

3

Completed RNA transcript

Unwound

DNA

RNA

transcript

Template strand of DNA

DNA

1 Initiation. After RNA polymerase binds to

the promoter, the DNA strands unwind, and

the polymerase initiates RNA synthesis at the

start point on the template strand.

2Elongation. The polymerase moves downstream, unwinding

the DNA and elongating the RNA transcript 5 3 . In

the wake of transcription, the DNA strands re-form a double helix.

3 Termination. Eventually, the RNA

transcript is released, and the

polymerase detaches from the DNA.

Dr. Sharma Jan,09

Elongation

RNApolymerase

Non-templatestrand of DNA

RNA nucleotides

3 end

C A E G C A A

U

T A G G T TA

AC

G

U

AT

CA

T C C A A TT

G

G

3

5

5

Newly madeRNA

Direction of transcription(“downstream”) Template

strand of DNA

Dr. Sharma Jan,09

RNA Polymerase Binding and RNA Polymerase Binding and Initiation of TranscriptionInitiation of Transcription

Promoters signal the initiation of RNA synthesisTranscription factors

Help eukaryotic RNA polymerase recognize promoter sequences

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

DNA

Pre-mRNA

mRNA

Ribosome

Polypeptide

T A T A AA AA T A T T T T

TATA box Start point TemplateDNA strand

53

35

Transcriptionfactors

53

35

Promoter

53

355

RNA polymerase IITranscription factors

RNA transcript

Transcription initiation complex

Eukaryotic promoters1

Several transcriptionfactors

2

Additional transcriptionfactors

3

Dr. Sharma Jan,09

Elongation of the RNA Elongation of the RNA StrandStrand

It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Dr. Sharma Jan,09

As RNA polymerase moves along the DNA

It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Elongation of the RNA StrandElongation of the RNA Strand

Dr. Sharma Jan,09

Termination of Termination of TranscriptionTranscription

The mechanisms of termination

Are different in prokaryotes and eukaryotes

Dr. Sharma Jan,09

Eukaryotic cells modify RNA Eukaryotic cells modify RNA after transcriptionafter transcription

Enzymes in the eukaryotic Enzymes in the eukaryotic nucleusnucleus

Modify pre-mRNA in specific ways Modify pre-mRNA in specific ways before the genetic messages are before the genetic messages are dispatched to the cytoplasmdispatched to the cytoplasm

Dr. Sharma Jan,09

Alteration of mRNA EndsAlteration of mRNA Ends

Each end of a pre-mRNA molecule is modified in a particular way

The 5 end receives a modified nucleotide capThe 3 end gets a poly-A tail

A modified guanine nucleotideadded to the 5 end

50 to 250 adenine nucleotidesadded to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail3 UTRStop codonStart codon

5 Cap 5 UTR

AAUAAA AAA…AAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

G P P P

53

Dr. Sharma Jan,09

Eukaryotic mRNAs are modified. At the 5' end, a cap is added consisting of a modified GTP (guanosine triphosphate). This occurs at the beginning of transcription. The 5' cap is used as a recognition signal for ribosomes to bind to the mRNA.• At the 3' end, a poly(A) tail of 150 or more adenine nucleotides is added. The tail plays a role in the stability of the mRNA.

Dr. Sharma Jan,09

Split Genes and RNA SplicingSplit Genes and RNA Splicing

RNA splicingRemoves introns and joins exons

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

5 Cap

ExonIntron

1

5

30 31

Exon Intron

104 105 146

Exon 3

Poly-A tail

Poly-A tail

Introns cut out andexons spliced together

Codingsegment

5 Cap

1 1463 UTR3 UTR

Pre-mRNA

mRNA

Dr. Sharma Jan,09

Is carried out by spliceosomes in some cases RNA transcript (pre-mRNA)

Exon 1 Intron Exon 2

Other proteinsProtein

snRNA

snRNPs

Spliceosome

Spliceosomecomponents

Cut-outintronmRNA

Exon 1 Exon 2

5

5

5

1

2

3

Dr. Sharma Jan,09

RibozymesRibozymes

RibozymesAre catalytic RNA molecules that function as

enzymes and can splice RNA

Dr. Sharma Jan,09

Translation is the RNA-directed synthesis of a polypeptide:

a closer look

Dr. Sharma Jan,09

Molecular Components of Molecular Components of TranslationTranslation

A cell translates an mRNA message into proteinWith the help of transfer RNA

(tRNA)

Dr. Sharma Jan,09

Translation: the basic conceptTranslation: the basic conceptTRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Polypeptide

Aminoacids

tRNA withamino acidattachedRibosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

A G C

A A A

CC

G

U G G U U U G G C

Codons5 3

Dr. Sharma Jan,09

Molecules of tRNA are not all identicalMolecules of tRNA are not all identicalEach carries a specific amino acid on one Each carries a specific amino acid on one

endend

Each has an anticodon on the otherEach has an anticodon on the other endend

Dr. Sharma Jan,09

The Structure and Function of TransferThe Structure and Function of Transfer

A tRNA moleculeConsists of a single RNA strand that is only about 80 nucleotides longIs roughly L-shaped

Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3’ end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.)

(a)

3

CCACGCUUAA

GACACCU*

GC

* *G U G U *CU

* G AGGU**A

*A

A GUC

AGACC*

C G A GA G G

G*

*GA

CUC*AUUUAGGCG5

Amino acidattachment site

Hydrogenbonds

Anticodon

A

Dr. Sharma Jan,09

A specific enzyme called an aminoacyl-tRNA A specific enzyme called an aminoacyl-tRNA synthetasesynthetaseJoins each amino acid to the correct tRNAJoins each amino acid to the correct tRNAAminoacyl-tRNA

synthetase (enzyme)

1. Active site binds theamino acid and ATP.

ATP loses two P groupsand joins amino acid as AMP.2

Pyrophosphate

Phosphates

3 AppropriatetRNA covalentlyBonds to aminoAcid, displacingAMP.

AMP

Activated amino acidis released by the enzyme.4

Aminoacyl tRNA(an “activatedamino acid”)

1. Enzyme+ Amino acid+ ATP

Enzyme-Aa-AMP complex +2Pi

2. Enzyme-Aa-AMP complex +tRNA

tRNA-Aa +AMP + Enzyme

Dr. Sharma Jan,09

Dr. Sharma Jan,09

(b) Three-dimensional structure

Symbol used in the presentation

Amino acidattachment site

Hydrogen bonds

AnticodonAnticodon

A A G

5

3

3 5

(c)

Dr. Sharma Jan,09

Ribosomes

Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis

Dr. Sharma Jan,09

The ribosomal subunitsThe ribosomal subunitsAre constructed of proteins and RNA molecules Are constructed of proteins and RNA molecules named ribosomal RNA or rRNAnamed ribosomal RNA or rRNA

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide Exit tunnelGrowingpolypeptide

tRNAmolecules

EP A

Largesubunit

Smallsubunit

mRNA

Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins.

53

Dr. Sharma Jan,09

We can divide translation into three stagesWe can divide translation into three stagesInitiation, Elongation, TerminationInitiation, Elongation, Termination

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams.

Dr. Sharma Jan,09

Amino end Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

mRNA

Codons

3

5

Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

(c)

Dr. Sharma Jan,09

Ribosome Association and Initiation of Ribosome Association and Initiation of TranslationTranslation

The initiation stage of translationBrings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosomeLarge

ribosomalsubunit

The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiationfactors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid.

2

Initiator tRNA

mRNA

mRNA binding site Smallribosomalsubunit

Translation initiation complex

P site

GDPGTP

Start codon

A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met).

1

MetMet

U A CA U G

E A

35

53

35 35

Dr. Sharma Jan,09

Elongation of the Polypeptide Chain-Elongation of the Polypeptide Chain-Amino acids are Amino acids are added one by one to the preceding amino acidadded one by one to the preceding amino acid

Amino endof polypeptide

mRNA

Ribosome ready fornext aminoacyl tRNA

E

P A

E

P A

E

P A

E

P A

GDPGTP

GTP

GDP

2

2

site site5

3

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysisof GTP increases the accuracy andefficiency of this step.

1

Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site.

2

Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs,bringing the next codon to be translated into the A site.

3

Dr. Sharma Jan,09

Termination of Translation-When the Termination of Translation-When the ribosome reaches a stop codon in the mRNAribosome reaches a stop codon in the mRNA

Release factor

Freepolypeptide

Stop codon(UAG, UAA, or UGA)

5

3 35

35

When a ribosome reaches a stop When a ribosome reaches a stop codon on mRNA, the A site of the codon on mRNA, the A site of the ribosome accepts a protein called ribosome accepts a protein called a release factor instead of tRNAa release factor instead of tRNA.

1 The release factor hydrolyzes The release factor hydrolyzes the bond between the tRNA in the bond between the tRNA in the P site and the last amino the P site and the last amino acid of the polypeptide chain. acid of the polypeptide chain. The polypeptide is thus freed The polypeptide is thus freed from the ribosome.from the ribosome.

2 3 The two ribosomal subunits The two ribosomal subunits and the other components of and the other components of the assembly dissociate.the assembly dissociate.

Dr. Sharma Jan,09

A number of ribosomes can translate a single A number of ribosomes can translate a single mRNA molecule simultaneously- mRNA molecule simultaneously- PolyribosomePolyribosome

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start of mRNA(5 end)

End of mRNA(3 end)

Polyribosome

An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

0.1 µm

(b)

Dr. Sharma Jan,09

Completing and Targeting the Functional Protein

Polypeptide chainsUndergo modifications after the translation process

Dr. Sharma Jan,09

Protein Folding and Post-Protein Folding and Post-Translational ModificationsTranslational Modifications

After translationProteins may be modified in ways that affect their three-dimensional shape

Dr. Sharma Jan,09

Targeting Polypeptides to Specific Targeting Polypeptides to Specific LocationsLocations

Two populations of ribosomes are evident in cells

Free and bound

Free ribosomes in the cytosolInitiate the synthesis of all proteins

Dr. Sharma Jan,09

• Proteins destined for the endomembrane Proteins destined for the endomembrane system or for secretionsystem or for secretion– Must be transported into the ERMust be transported into the ER– Have signal peptides to which a signal-recognition Have signal peptides to which a signal-recognition

particle (SRP) binds, enabling the translation particle (SRP) binds, enabling the translation ribosome to bind to the ERribosome to bind to the ER

Dr. Sharma Jan,09

The signal mechanism for The signal mechanism for targeting proteins to the ERtargeting proteins to the ER

Figure 17.21

Ribosome

mRNASignalpeptide

Signal-recognitionparticle(SRP) SRP

receptorprotein

Translocationcomplex

CYTOSOL

Signalpeptideremoved

ERmembrane

Protein

ERLUMEN

PolypeptidePolypeptidesynthesis beginssynthesis beginson a freeon a freeribosome inribosome inthe cytosol.the cytosol.

1

An SRP binds An SRP binds to the signal to the signal peptide, halting peptide, halting synthesissynthesismomentarily.momentarily.

2 The SRP binds to aThe SRP binds to areceptor protein in the ERreceptor protein in the ERmembrane. This receptormembrane. This receptoris part of a protein complexis part of a protein complex(a translocation complex)(a translocation complex)that has a membrane porethat has a membrane poreand a signal-cleaving enzymeand a signal-cleaving enzyme.

3 The SRP leaves, andThe SRP leaves, andthe polypeptide resumesthe polypeptide resumesgrowing, meanwhilegrowing, meanwhiletranslocating across thetranslocating across themembrane. (The signalmembrane. (The signalpeptide stays attachedpeptide stays attachedto the membrane.)to the membrane.)

4

The signal-The signal-cleaving cleaving enzymeenzymecuts off thecuts off thesignal peptidesignal peptide.

5 The rest ofThe rest ofthe completedthe completedpolypeptide leaves polypeptide leaves the ribosome andthe ribosome andfolds into its finalfolds into its finalconformation.conformation.

6