PROTEIN SYNTHESISPROTEIN SYNTHESIS

HOW GENES ARE HOW GENES ARE EXPRESSEDEXPRESSED

BEADLE AND TATUM-1930’SBEADLE AND TATUM-1930’SOne Gene-One Enzyme HypothesisOne Gene-One Enzyme Hypothesis

PROTEIN SYNTHESISPROTEIN SYNTHESIS

GENE (DNA) GENE (DNA) TRANSCRIPTION TRANSCRIPTION TRANSLATION TRANSLATION PROTEIN PROTEIN

– DNA directs the synthesis of RNA, which is DNA directs the synthesis of RNA, which is the link between genes and proteinsthe link between genes and proteins

GENETIC CODEGENETIC CODE

Triplet codeTriplet code– Three nucleotide sequence codes for an Three nucleotide sequence codes for an

amino acid amino acid Called a “codon” in mRNA; “anticodon” in tRNACalled a “codon” in mRNA; “anticodon” in tRNA

– 64 possible combinations code for 20 amino 64 possible combinations code for 20 amino acidsacids

There are also “stop” and “start” codesThere are also “stop” and “start” codes

There is redundancy in the codeThere is redundancy in the code

TRANSCRIPTIONTRANSCRIPTION

Process varies between prokaryotes and Process varies between prokaryotes and eukaryoteseukaryotes– HOW??HOW??

Transcription is DNA-directed synthesis of Transcription is DNA-directed synthesis of mRNAmRNA

TRANSCRIPTIONTRANSCRIPTION

Transcription has 3 stagesTranscription has 3 stages– InitiationInitiation– ElongationElongation– terminationtermination

Transcription: InitiationTranscription: Initiation

One strand of DNA is used as a templateOne strand of DNA is used as a template– RNA polymerase: attaches to the promoter RNA polymerase: attaches to the promoter

site and pulls apart the DNA strandssite and pulls apart the DNA strands– Transcription occurs in the 5’ Transcription occurs in the 5’ 3’ direction 3’ direction– ComplementaryComplementary nucleotide bases are added nucleotide bases are added

REMEMBER: there is no “T” in RNA; instead, “A” REMEMBER: there is no “T” in RNA; instead, “A” pairs with “U”pairs with “U”

TRANSCRIPTION: ELONGATIONTRANSCRIPTION: ELONGATION

Nucleotides are added to the 3’ end of the Nucleotides are added to the 3’ end of the growing mRNA strandgrowing mRNA strand

About 60 nucleotides/sec are addedAbout 60 nucleotides/sec are added

Length of the primary transcript varies Length of the primary transcript varies depending on protein being produceddepending on protein being produced

TRANSCRIPTION: ELONGATIONTRANSCRIPTION: ELONGATION

TRANSCRIPTION: TERMINATIONTRANSCRIPTION: TERMINATION

RNA polymerase transcribes a terminator RNA polymerase transcribes a terminator sequence in the DNAsequence in the DNA

Details of actual events in termination are Details of actual events in termination are still not entirely clearstill not entirely clear

After termination, transcript is called “PRE-After termination, transcript is called “PRE-mRNAmRNA– Modification of mRNA occurs after terminationModification of mRNA occurs after termination

mRNA ModificationmRNA Modification

5’ cap is added5’ cap is added– Modified guanine nucleotideModified guanine nucleotide– Protects the transcript from being broken Protects the transcript from being broken

down by enzymesdown by enzymes– Provides a signal for ribosomal attachmentProvides a signal for ribosomal attachment

Poly-A tail is added to the 3’ endPoly-A tail is added to the 3’ end– 50 – 250 adenine nucleotides50 – 250 adenine nucleotides– Inhibits breakdown,assists in attachment, and Inhibits breakdown,assists in attachment, and

aids in export out of nucleusaids in export out of nucleus

mRNA ModificationmRNA Modification

RNA splicingRNA splicing– Noncoding regions of transcript are removedNoncoding regions of transcript are removed

Called INTRONSCalled INTRONS

– Coding sections are called EXONSCoding sections are called EXONS

mRNA processing: spliceosomesmRNA processing: spliceosomesWhat makes up a spliceosome?What makes up a spliceosome?

mRNA processingmRNA processing

Why are some parts of mRNA non-Why are some parts of mRNA non-coding?coding?

In other words, why are there introns?In other words, why are there introns?

– Perhaps allows flexibility in coding for different Perhaps allows flexibility in coding for different proteins, by simply splicing differentlyproteins, by simply splicing differently

Called alternative RNA splicingCalled alternative RNA splicing

TRANSLATION: FROM mRNA to TRANSLATION: FROM mRNA to PROTEINPROTEIN

mRNA leaves the nucleus of the cell through mRNA leaves the nucleus of the cell through nuclear poresnuclear pores

Translation occurs in the cytoplasm on a Translation occurs in the cytoplasm on a ribosomeribosome

Translation requires tRNATranslation requires tRNA– Each tRNA has a nucleotide sequence called an Each tRNA has a nucleotide sequence called an

ANTICODON that is complementary to the mRNA ANTICODON that is complementary to the mRNA codecode

– tRNA is made in the nucleus; used repeatedly in the tRNA is made in the nucleus; used repeatedly in the cytoplasmcytoplasm

tRNAtRNA

tRNAtRNA

Each tRNA contains a 3-letter code that Each tRNA contains a 3-letter code that codes for an amino acidcodes for an amino acid– What is WOBBLE?What is WOBBLE?

Variability in coding of the 3Variability in coding of the 3rdrd base pairing in the base pairing in the tRNAtRNA

TRANSLATIONTRANSLATIONEach tRNA must first pick up its correct Each tRNA must first pick up its correct amino acidamino acid– Joined by aminoacyl-tRNA synthetaseJoined by aminoacyl-tRNA synthetase– Now called an activated amino acidNow called an activated amino acid– Also called “tRNA charging”Also called “tRNA charging”

TRANSLATIONTRANSLATION

The process occurs at a ribosomeThe process occurs at a ribosome

Ribosomes-made up of proteins and rRNARibosomes-made up of proteins and rRNA

RibosomeRibosome

P site (peptidyl-tRNA)-holds the tRNA P site (peptidyl-tRNA)-holds the tRNA carrying the growing polypeptide chaincarrying the growing polypeptide chain

A site (aminoacyl-tRNA site) holds the A site (aminoacyl-tRNA site) holds the tRNA bringing the next amino acidtRNA bringing the next amino acid

E site (exit site)-where “used” tRNA’s E site (exit site)-where “used” tRNA’s leave the ribosomeleave the ribosome

TRANSLATIONTRANSLATION

Occurs in 3 stagesOccurs in 3 stages– InitiationInitiation– ElongationElongation– TerminationTermination

Energy requiring processEnergy requiring process– Hydrolysis of GTPHydrolysis of GTP

TRANSLATION: INITIATIONTRANSLATION: INITIATION

Coming together of mRNA and a tRNA Coming together of mRNA and a tRNA carrying the first amino acidcarrying the first amino acid– Complex processComplex process– Small ribosomal subunit binds to mRNA and Small ribosomal subunit binds to mRNA and

initiator tRNA; then large ribosomal subunit initiator tRNA; then large ribosomal subunit attaches forming the TRANSLATION attaches forming the TRANSLATION INITIATION COMPLEXINITIATION COMPLEX

TRANSLATION: INITIATIONTRANSLATION: INITIATION

TRANSLATION: ELONGATIONTRANSLATION: ELONGATION

Amino acids are added sequentially to the Amino acids are added sequentially to the growing polypeptide chain, requiring help growing polypeptide chain, requiring help of elongation factorsof elongation factors

Occurs in 3 stepsOccurs in 3 steps

TRANSLATION: ELONGATIONTRANSLATION: ELONGATIONCodon recognitionCodon recognition

New tRNA is helped into A siteNew tRNA is helped into A site

Peptide bond formationPeptide bond formationrRNA (ribozyme) catalyzes a peptide bond forming rRNA (ribozyme) catalyzes a peptide bond forming between the amino acid in the P site and the one between the amino acid in the P site and the one in the A site. Growing polypeptide chain is now in in the A site. Growing polypeptide chain is now in the A sitethe A site

TranslocationTranslocationAmino acid in A site is moved to P siteAmino acid in A site is moved to P site

tRNA in P site is moved to E sitetRNA in P site is moved to E site

TRANSLATION: ELONGATIONTRANSLATION: ELONGATION

TRANSLATION: TERMINATIONTRANSLATION: TERMINATION

Elongation continues until stop codon in Elongation continues until stop codon in the mRNA reaches the A sitethe mRNA reaches the A site

UAA, UAG, UGAUAA, UAG, UGA

Release factor binds to the stop codon in Release factor binds to the stop codon in the A sitethe A site– Water molecule is added to the polypeptide Water molecule is added to the polypeptide

chain, thus releasing the polypeptide chain chain, thus releasing the polypeptide chain from the ribosomefrom the ribosome

TRANSLATION: TERMINATIONTRANSLATION: TERMINATION

PolyribosomesPolyribosomes

Help to speed up process of protein Help to speed up process of protein formation, as mRNA can be translated formation, as mRNA can be translated simultaneously by multiple ribosomessimultaneously by multiple ribosomes

Posttranslational ModificationPosttranslational Modification

After release from ribosome, polypeptide After release from ribosome, polypeptide may require some changes before it is a may require some changes before it is a fully functional proteinfully functional protein– Sugars, lipids, phosphates addedSugars, lipids, phosphates added– Several polypeptides may be joined togetherSeveral polypeptides may be joined together

Protein Synthesis: A summaryProtein Synthesis: A summary