GIO: L4 02-05 FROM DNA TO PROTEIN

WHAT IS THE DIFFERENCE BETWEEN RNA VS. DNA?

• RNA CONTAINS A HYDROXYL (-OH) GROUP ON THE 2’ CARBON OF THE SUGAR

• RNA CONTAINS URACIL INSTEAD OF THYMINE

  • U PAIRS WITH A

• RNA IS SINGLE-STRANDED

RNA CAN…(4 FLASHCARDS)

• CAN FOLD INTO STABLE 3D STRUCTURES

  • HYDROGEN BONDS STABILIZE RNA STRUCTURES

• CAN STORE INFORMATION AND COPY BY ITSELF

  • RNA STRANDS=ANTI-PARALLEL

  • FOR EXAMPLE: RNA VIRUSES STORE THEIR GENETIC INFORMATION AS RNA

  • MODEL FOR SELF-TEMPLATED RNA REPLICATION

  • RNA IS THOUGHT TO HAVE PRECEDED DNA AND PROTEINS IN EVOLUTION

• CAN HAVE CATALYTIC ACTIVITY

  • RIBOZYMES: RNA MOLECULES WITH CATALYTIC ACTIVITIES

WHAT ARE THE TYPES OF RNA PRODUCED IN CELLS?

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WHAT IS TRANSCRIPTION?

• DNA—>RNA

• THE RATES OF BOTH TRANSCRIPTION AND TRANSLATION CAN DIFFER BETWEEN GENES

• ALL RNA MOLECULES (EXCEPT IN SOME VIRUSES) ARE DERIVED FROM INFORMATION PERMANENTLY STORED IN DNA

• DURING REPLICATION, THE ENTIRE GENOME IS USUALLY COPIED

• DURING TRANSCRIPTION, ONLY PARTICULAR GENES OR GROUPS OF GENES ARE TRANSCRIBED AT ANY ONE TIME (SOME PORTIONS OF THE DNA GENOME ARE NEVER TRANSCRIBED)

• ONLY ONE DNA STRAND SERVES AS A TEMPLATE FOR TRANSCRIPTION

• DNA CONTAINS

  • A CODING STAND

  • A TEMPLATE STRAND

• THE SEQUENCE OF THE RNA TRANSCRIPT MATCHES THE SEQUENCE OF THE CODING STRAND

• THE TEMPLATE STRAND IS COMPLEMENTARY TO THE RNA TRANSCRIPT

• *REMEMBER-GENES CAN BE ORIENTED IN EITHER DIRECTION ON THE DNA

• REPLICATION IS CARRIED OUT BY DNA POLYMERASES

• TRANSCRIPTION IS CARRIED OUT BY RNA POLYMERASES

DNA IS TRANSCRIBED INTO…

• RNA BY ENZYMES CALLED RNA POLYMERASES

• SYNTHESIS IS 5’—>3’

• SYNTHESIS IS DE NOVO

• NO PRIMER IS REQUIRED

• THE TEMPLATE STRAND OF THE DNA IS READ BY RNA POLYMERASE TO PRODUCE THE NEWLY SYNTHESIZED RNA TRANCRIPT

• RNA POLYMERASE CATALYZES THE FORMATION OF PHOSPHODIESTER BONDS, ELONGATING THE TRANSCRIPT ONE NUCLEOTIDE AT A TIME

• THE ENERGY FOR PHOSPHODIESTER BOND FORMATION COMES FROM THE PHOSPHOANHYDRIDE BOND IN THE ATP, GTP, CTP, OR UTP BEING INCORPORATED

MANY MOLECULES OF RNA POLYMERASE….

• CAN SIMULTANEOUSLY TRANSCRIBE THE SAME GENE

• DIRECTION?

• >1000 TRANSCRIPT COPIES CAN BE SYNTHESIZED IN 1 HOUR. HOWEVER, MOST GENES ARE NOT THAT HIGHLY TRANSCRIBED

WHAT ARE THE 3 MAJOR STAGES OF TRANSCRIPTION?

1) INITIATION

2) ELONGATION

3) TERMINATION

• RNA SYNTHESIS IS DE NOVO

• NO PRIMER. IS REQUIRED!

• HOW DOES THE RNA POLYMERASE KNOW WHERE TO INITIATE TRANSCRIPTION?

• IN EUKARYOTES, TERMINATION IS COUPLED TO RNA PROCESSING

WHERE IS TRANSCRIPTION INITIATED?

• IT IS INITIATED AT PROMOTERS

• RNA POLYMERASES RECOGNIZE AND BIND TO SPECIFIC DNA SEQUENCES CALLED PROMOTERS

• THE 1ST NUCLEOTIDE TRANSCRIBED IS DESIGNATED +1

• THE PROMOTER SEQUENCE IS ASYMMETRIC, ALLOWING RNAP TO BIND ONLY IN THE CORRECT ORIENTATION

• NOTE THAT THE PROMOTER SEQUENCE ITSELF IS NOT TRANSCRIBED INTO RNA

WHAT IS TRANSCRIPTION IN BACTERIA?

• RNA POLYMERASE (RNAP) SCANS DNA AND BINDS TIGHTLY TO PROMOTORS

• PROMOTOR RECOGNITION AND BINDING REQUIRES A SIGMA FACTOR

• TRANSCRIPTION INITIATES AT THE START SITE (+1)

• RNAP TRANSITIONS TO ELONGATION WITH RELEASE OF SIGMA FACTOR

• ELONGATION CONTINUES UNTIL RNAP ENCOUNTERS A TERMINATION SIGNAL

• RNAP RELEASES BOTH THE DNA TEMPLATE AND THE RNA TRANSCRIPT

• FREE RNAP ASSOCIATES WITH A FREE SIGMA FACTOR TO BEGIN AGAIN

• ALTHOUGH THE PROMOTER SEQUENCE IS NOT TRANSCRIBED INTO RNA, THE TERMINATION SEQUENCE IS PART OF THE RNA TRANSCRIPT

GENES CAN BE TRANSCRIBED…

• FROM EITHER STRAND OF DNA

• THE PROMOTER ORIENTATION DETERMINES WHICH STRAND IS TRANSCRIBED

• RNA POLYMERASE ALWAYS MOVES IN THE 3’—→5’ DIRECTION WITH RESPECT TO THE TEMPLATE DNA STRAND

WHAT IS TRANSCRIPTION IN EUKARYOTES LIKE?

• MUCH MORE COMPLEX

• EUKARYOTES HAVE 3 DIFFERENT RNA POLYMERASES

WHAT IS REQUIRED TO INITIATE TRANSCRIPTION IN EUKARYOTES?

• A SET OF GENERAL TRANSCRIPTION FACTORS

• GENERAL TRANSCRIPTION FACTORS ARE ACCESSORY PROTEINS THAT POSITION THE POLYMERASE AND PULL APART THE DNA

• THESE FACTORS + RNA POLYMERASE II MAKE UP THE TRANSCRIPTION+INITIATION COMPLEX

• MANY EUKARYOTIC PROMOTERS CONTAIN A RECOGNITION SEQUENCE CALLED A TATA BOX ABOUT 30 NUCLEOTIDES UPSTREAM OF THE START SITE

• TBP (TATA-BINDING PROTEIN), A SUBUNIT. OF TFIID, RECOGNIZES THE TATA BOX

• TFIIH UNWINDS THE HELIX AND PHOSPHORYLATES THE TAIL OF RNA POLYMERASE II (RNAPII)

• PHOSPHORYLATED RNAP II IS RELEASED TO BEGIN TRANSCRIPTION ELONGATION

EUKARYOTIC PROMOTERS CONTAIN…

• SEQUENCES THAT PROMOTE BINDING OF GENERAL TRANSCRIPTION FACTORS

• THE PROMOTER SEQUENCE IS ASYMMETRIC, ALLOWING RNAP TO BIND ONLY IN THE CORRECT ORIENTATION

WHAT IS TRANSCRIPTION: ELONGATION?

• PHOSPHORYLATION ON RNAPII BY TFIIH PROMOTES RELEASE OF THE GENERAL TRANSCRIPTION FACTORS

• ELONGATION FACTORS ALLOW RNAPII TO MOVE THROUGH DNA THAT IS PACKAGED INTO NUCLEOSOMES

WHAT IS PROCESSED IN THE NUCLEUS?

• EUKARYOTIC RNAS ARE PROCESSED IN THE NUCLEUS

• IN PROKARYOTES, RIBOSOMES IMMEDIATELY ATTACH TO THE 5’ ENDS OF TRANSCRIPTS TO BEGIN TRANSLATION

• EUKARYOTIC RNAS MUST BE TRANSPORTED OUT OF THE NUCLEUS THROUGH NUCLEAR PORES (ARROWS)

• FIRST, THE RNAS MUST BE PROCESSED…

WHAT IS EUKARYOTIC RNA PROCESSING?

3 RNA PROCESSING STEPS:

1. CAPPING

2. SPLICING

3. POLYADENYLATION

• RNA PROCESSING FACTORS ASSEMBLE ON THE PHOSPHORYLATED TAIL OF RNAP11

• RNA PROCESSING IS CO-TRANSCRIPTIONAL

WHAT IS 5’ CAPPING AND 3’ POLYADENYLATION?

• CAPPING MODIFIES THE 5’ END OF THE MRNA

  • THE CAP IS MODIFIED GUANINE NUCLEOTIDE

  • CAPPING OCCURS AFTER ~25 NTS HAVE BEEN TRANSCRIBED

• POLYADENYLATION MODIFIES THE 3’ END

  • THE 3’ END OF THE MRNA IS FIRST TRIMMED

  • A SECOND ENZYME ADDS ADENINE(A) REPEATS TO THE 3’ END

  • THE RESULT IS POLY-A TAIL

• IMPORTANT FOR:

  • EXPORT

  • STABILITY

  • TRANSLATION

WHAT DO MOST EUKARYOTIC GENES CONTAIN?

• INTRONS!

• EXONS: CODING (EXPRESSED) SEQUENCES ARE SPLICED TOGETHER

• INTRONS: NONCODING (INTERVENING) SEQUENCES ARE REMOVED DURING SPLICING

WHAT ARE REMOVED BY SPLICING?

• INTRONS!

• BEFORE SPLICING, THE RNA IS CONSIDERED A PRE-MRNA

• SPLICING IS PERFORMED BY SPLICING MACHINERY THAT RECOGNIZES SPECIFIC SEQUENCES IN THE PRE-MRNA

• SPLICING INVOLVES 2 TRANSESTERIFICATION REACTIONS. THE 2’ OH OF THE BRANCH-POINT ADENOSINE (A) ATTACKS THE PHOPHODIESTER BOND AT THE 5’ SPLICE STRUCTURE

• THE 3’ OH AT THE 3’ END OF THE EXON ATTACKS THE PHOSPHODIESTER BOND AT THE 3’ END OF THE INTRON TO JOIN THE EXONS

• THE LARIAT STRUCTURE IS RELEASED

WHAT IS THE SPLICING MACHINERY?

• SPLICING IS CARRIED OUT BY THE SPLICEOSOME

• snRNPs (SMALL NUCLEAR RIBONUCLEOPROTEINS) ARE FUNCTIONAL UNITS OF THE SPLICEOSOME

• snRNPs CONTAIN snRNAs THAT FORM THE ACTIVE SITE OF THE SPLICEOSOME

  • snRNA=SMALL NUCLEAR RNA

• snRNAs RECOGNIZE SPLICE SITE SEQUENCES VIA COMPLEMENTARY BASE PAIRING

WHAT ALLOWS DIFFERENT PROTEINS TO BE PRODUCED FROM A SINGLE GENE?

• ALTERNATIVE SPLICING!

WHAT ARE EXPORTED TO THE CYTOPLASM THROUGH NUCLEAR PORES?

• MATURE MRNAS

• ONLY CORRECTLY PROCESSED MRNAS ARE EXPORTED. THESE RNAS ARE BOUND TO:

  1. CAP-BINDING PROTEIN

  2. POLY-A-BINDING PROTEIN

  3. EXON JUNCTION COMPLEX

• NUCLEAR PORE COMPLEXES CONNECT THE NUCLEOPLASM WITH THE CYTOSOL

• MRNAS ARE TRANSLATED IN THE CYTOSOL AND EVENTUALLY DEGRADED

• THE LIFETIME OF A EUKARYOTIC MRNA IS VARIABLE (30 MIN-10 HOURS)

• THE 3’UTR CONTRIBUTES TO THE STABILITY OF THE MRNA

WHAT IS MORE COMPLEX IN EUKARYOTES THAN IN PROKARYOTES?

• GENE EXPRESSION!

WHAT IS THE ADAPTOR MOLECULE?

• TRNA !

• A SINGLE AMINO ACID IS COVALENTLY ATTACHED TO THE 3’ END

• THE ANTICODON PAIRS WITH THE MRNA

WHAT IS THE GENETIC CODE?

• IS A NONOVERLAPPING TRIPLET CODE

• FOR 20 AMINO ACIDS, AT LEAST 3 NUCLEOTIDES ARE NECESSARY PER CODON

• BECAUSE THE CODE IS NONOVERLAPPING, EACH FRAME GIVES A DIFFERENT SET OF TRIPLETS

WHAT IS THE READING FRAME?

• IN HOW MANY WAYS CAN WE READ THIS?

WHAT DOES THE GENETIC CODE DEFINE?

• THE GENETIC CODE DEFINES THE RULES BY WHICH THE NUCLEOTIDE SEQUENCE OF A GENE IS TRANSLATED INTO THE AMINO ACID SEQUENCE OF THE PROTEIN

• THE GENETIC CODE IS A NONOVERLAPPING TRIPLET CODE-EACH TRIPLET IS CALLED A CODON

• CODONS CODE FOR AMINO ACIDS, EXCEPT FOR THREE STOP CODONS

• THERE ARE 64 CODONS BUT ONLY 20 AMINO ACIDS-THE CODE IS REDUNDANT

• THERE IS ONLY 1 CODON THAT START ALL PROTEINS: AUG-THE START CODON

HOW DOES THE TRNA RECOGNIZE THE SPECIFIC AMINO ACID?

• REMEMBER THIS?

WHAT IS THE GENETIC CODE TRANSLATED BY?

• BY AMINOACYL-TRNA SYNTHESASES AND TRNAS!

• EACH AMINO ACID HAS A DIFFERENT SYNTHETASE ENZYME-A PROTEIN! (20 IN TOTAL)

• SYNTHETASES RECOGNIZE SEQUENCES IN THE ANTICODON AND THE AMINO ACID-ACCEPTING ARM

• TRNAS COVALENTLY LINKED TO AMINO ACIDS AT THEIR 3’ ENDS ARE CHARGED

WHAT MESSAGE IS DECODED ON RIBOSOMES?

• THE MRNA MESSAGE

• MOST EUKARYOTIC CELLS HAVE MILLIONS OF RIBOSOMES IN THE CYTOPLASM

• THE RIBOSOME IS A LARGE COMPLEX OF rRNAS AND RIBOSOMAL PROTEINS

• THE RIBOSOME HAS TWO SUBUNITS: A LARGE SUBUNIT AND A SMALL SUBUNIT

• 4 STAGES OF TRANSLATION:

  1. CHARGING

  2. INITIATION

  3. ELONGATION

  4. TERMINATION

WHAT IS THE EUKARYOTIC RIBOSOME?

• SMALL SUBUNIT

  • BINDS THE FIRST MRNA

  • MATCHES TRNAS TO CODONS

• LARGE SUBUNIT

  • CATALYZES PEPTIDE BOND FORMATION

• INITIATION: THE SUBUNITS COME TOGETHER NEAR THE 5’ END OF THE MRNA

• ELONGATION: THE MRNA IS PULLED THROUGH THE RIBOSOME AND TRANSLATED ONE CODON AT A TIME USING TRNAS AS ADAPTORS

• TERMINATION: THE SUBUNITS SEPARATE WHEN TRANSLATION IS COMPLETE

• PROKARYOTIC RIBOSOMES ALSO HAVE A SMALL AND A LARGE SUBUNIT

• RATES:

  • 2 AMINO ACIDS/SEC IN EUKARYOTES

  • 20 AMINO ACIDS/SEC IN PROKARYOTES

• EACH RIBOSOME HAS:

  • 1 MRNA BINDING SITE

  • 3 TRNA BINIDNG SITES

    • A=AMINOACYL-TRNA SITE

    • P=PEPTIDYL-TRNA SITE

    • E=EXIT SITE

WHAT IS THE INITIATION OF TRANSLATION?

• TRANSLATION BEGINS WITH AN AUG CODON AND ESTABLISHES A READING FRAME

• INITIATOR TRNA IS A SPECIALLY CHARGED TRNA THAT CARRIES METHIONINE

• INITIATOR TRNA AND INITIATION FACTORS ARE LOADED INTO THE P SITE OF THE SMALL SUBUNIT

• THE COMPLEX BINDS TO THE 5’ CAP OF THE MRNA

• THE COMPLEX MOVES ALONG THE MRNA “SCANNING” FOR THE FIRST AUG CODON

• THE LARGE SUBUIT BINDS, AND THE INITIATION FACTORS DISSOCIATE

• A CHARGED TRNA BINDS IN THE A SITE, AND IF THE CODON-ANTICODON BASE PAIRING IS CORRECT, PEPTIDE BOND FORMATION OCCURS

WHAT IS ELONGATION?

• ELONGATION IS A 4-STEP CYCLE

  1. CHARGED TRNAS ENTER THE A SITE AND BASE PAIR WITH THE CODON ON THE MRNA

  2. PEPTIDE BOND FORMATION OCCURS BETWEEN THE NEW AMINO ACID AND THE CHAIN HELD BY THE TRNA IN THE P SITE

  3. THE LARGE SUBUNIT SHIFTS BY ONE CODON, MOVING THE UNCHARGED TRNA TO THE E SITE

  4. THE SMALL SUBUNIT TRANSLOCATES AND THE UNCHARGED TRNAS IS RELEASED

• THIS CYCLE IS REPEATED UNTIL A STOP CODON IS REACHED

  • MRNA IS READ 5’ —> 3’

  • PROTEIN IS MADE N—>C

WHAT IS THE TERMINATION OF TRANSLATION?

• THE TRANSLATION IS TERMINATED AT A STOP CODON

  • UAA, UAG, UGA

• RELEASE FACTOR BINDS THE A SITE AT STOP CODONS INSTEAD OF A CHARGED TRNA

• WATER IS ADDED TO THE POLYPEPTIDE INSTEAD OF AN AMINO ACID

• THE SYNTHESIZED POLYPEPTIDE IS RELEASED AND THE RIBOSOME DISSOCIATES

• PROTEINS ARE SYNTHESIZED ON POLYRIBOSOMES THROUGH MULTIPLE ROUNDS OF INITIATION, ELONGATION, AND TERMINATION

WHAT IS POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS?

• POST-TRANSLATIONAL MODIFICATIONS INCLUDE

  1. METHYLATION

  2. ACETYLATION

  3. PHOSPHORYLATION

  4. UBIQUITINATION

  5. GLYCOSYLATION

• MODIFICATIONS CAN AFFECT BINDING TO CO-FACTORS, STABILITY, AND SUBCELLULAR LOCALIZATION

WHAT IS REGULATION OF PROTEIN DEGRADATION?

• PROTEOLYSIS: THE BREAKDOWN OF PROTEINS INTO THEIR CONSTITUENT AMINO ACIDS

• PROTEASES: CUT PEPTIDE BONDS

• PROTEASOME: A MULTI-SUBUNIT MACHINE THAT DEGRADES PROTEINS

• PROTEINS TO BE DEGRADED ARE MARKED BY THE COVALENT ADDITION OF UBIQUITIN

WHAT IS THE OVERVIEW OF EUKARYOTIC GENE EXPRESSION?

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