Transcript protein synthesis overview

PROTEIN SYNTHESIS
OVERVIEW
• RNA LINKS DNA’S GENETIC INSTRUCTIONS
FOR MAKING PROTINS TO THE PROCESS OF
PROTEIN SYNTHESIS
• RNA COPIES (TRANSCRIBES) THE MESSAGE
FROM DNA AND THEN TRANSLATES THAT
MESSAGE INTO A PROTEIN
• THE LINEAR SEQUENCE OF NUCLEOTIDES
IN DNA DETERMINES THE LINEAR
SEQUENCE OF AMINO ACIDS IN A PROTEIN
• 3 STEPS: TRANSCRIPTION, RNA
PROCESSSING, TRANSLATION
VIDEO: PROTEIN SYNTHESIS OVERVIEW
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NUCLEOTIDE TRIPLET CODONS
• CODON = A THREE NUCLEOTIDE
SEQUENCE IN mRNA THAT SPECIFIES
WHICH AMINO ACID WILL BE ADDED TO
THE GROWING POLYPEPTIDE CHAIN
• READING FRAME = THE CORRECT
GROUPING OF ADJACENT NUCLEOTIDE
TRIPLETS INTO CODONS THAT ARE IN
THE CORRECT SEQUENCE ON THE mRNA
THE TRIPLET CODON
DICTIONARY OF THE GENETIC CODE
TRANSCRIPTION
• TRANSCRIPTION = THE SYNTHESIS OF RNA
USING DNA AS A TEMPLATE
• TRANSCRIPTION OF MESSENGER (mRNA) FROM
TEMPLATE DNA IS CATALYZED BY RNA
POLYMERASES WHICH:
– 1) SEPARATE THE TWO DNA STRAND AND LINK RNA
NUCLEOTIDES AS THEY BASE-PAIR ALONG THE DNA
TEMPLATE
– 2) ADD NUCLEOTIDES ONLY TO THE 3’ END; THUS,
mRNA MOLECULES GROW IN THE 5’ TO 3’
DIRECTIONS
– ** TRANSCRIPTION OCCURS IN 3 STAGES: A)
POLYMERASE BINDING AND INITIATION; B)
ELONGATION; AND C) TERMINATION
RNA POLYMERASE
BINDING AND INIATION
• RNA POLYMERASES BIND TO DNA AT REGIONS
CALLED PROMOTERS
• PROMOTERS = REGION OF DNA THAT INCLUDES
THE SITE WERE RNA POLYMERASE BINDS AND
WHERE TRANSCRIPTION BEGINS; ABOUT 100
NUCLEOTIDES LONG
• TRANSCRIPTION FACTORS = DNA BINDING
PROTEINS WHICH BIND TO SPECIFIC DNA
NUCLEOTIDE SEQUENCES AT THE PROMOTER
AND HELP RNA POLYMERASE RECOGNIZE AND
BIND TO THE PROMOTER REGION SO
TRANSCRIPTION CAN BEGIN
ELONGATION OF RNA
• ONCE TRANSCRIPTION BEGINS, RNA
POLYMERASE II MOVES ALONG DNA AND
PERFORMS TWO FUNCTIONS:
• 1) IT UNTWISTS AND OPENS A SHORT
SEGMENT OF DNA EXPOSING ABOUT TEN
NUCLEOTIDE BASES; ONE OF THE EXPOSED
DN STRAND IS THE TEMPLATE FOR BASEPAIRING WITH RNA NUCLEOTIDES
• 2) IT LINKS INCOMING RNA NUCLEOTIDES TO
THE 3’ END OF THE STRAND, THUS RNA
GROWS ONE NUCLEOTIDE AT A TIME IN THE 5’
TO 3’ DIRECTION
ELONGATION OF RNA
• DURING TRANSCRIPTION, mRNA GROWS
ABOUT 30 TO 60 NUCLEOTIDES PER
SECOND. AS THE mRNA STRAND
ELONGATES:
– 1) IT PEELS AWAY FROM ITS DNA TEMPLATE
– 2) THE NONTEMPLATE STRAND OF DNA REFORMS A DNA-DNA DOUBLE HELIX BY
PAIRING WITH TEMPLATE STRAND
– 3) SEVERAL RNA POLYMERASE II MOLECULES
CAN SIMULANEOUSLY TRANSCRIBE THE
SAME GENE; THUS, CELLS CAN PRODUCE
PARTICULAR PROTEINS IN LARGE AMTS.
TERMINATION OF
TRANSCRIPTION
• TRANSCRIPTION PROCEEDS UNTIL RNA
POLYMERASE TRANSCRIBES A DNA
SEQUENCE CALLED A TERMINATOR.
THIS SIGNALS THE END OF THE
TRANSCRIPTION PROCESS
TRANSCRIPTION VIDEO
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STAGES OF TRANSCRIPTION
RNA PROCESSING
• RNA TRANSCRIPTS IN EUKARYOTES
ARE PROCESSED BEFORE LEAVING THE
NUCLEUS TO YIELD FUNCTIONAL
mRNA. THIS PROCESSING CAN BE DONE
IN 2 WAYS:
• 1) COVALENT ALTERATION OF BOTH THE
3’ AND 5’ ENDS
• 2) REMOVAL OF INTERVENING
SEQUENCES
RNA PROCESSING
• PRIMARY TRANSCRIPT = GENERAL
TERM FOR INITIAL RNA
TRANSCRIBED BY DNA
• PRE-mRNA = PRIMARY TRANSCRIPT
THAT WILL BE PROCESSED TO
FUNCTIONAL MRNA
ADDITION OF 5’ CAP
• 5’ CAP = MODIFIED GUANINE NUCLEOTIDE
THAT IS ADDED TO THE 5’ END OF MRNA
SHORTLY AFTER TRANSCRIPTION BEGINS. IT
HAS 2 IMPORTANT FUNCTIONS:
• 1) PROTECTS THE GROWING mRNA FROM
DEGRADATION BY HYDROLYTIC ENZYMES
• 2) HELPS SMALL RIBOSOMAL SUBUNITS
RECOGNIZE THE ATTACHMENT SITE ON
mRNA’S 5’ END. A LEADER SEGMENT OF mRNA
MAY ALSO BE PART OF THE RIBOSOME
RECOGNITION SIGNAL
• LEADER SEQUENCE = NONCODING
(UNTRANSLATED) SEQUENCE OF mRNA FROM
THE 5’ END TO THE START CODON
ADDITION OF 5’ CAP AND POLY(A) TAIL
ADDITION OF POLY(A) TAIL
• THE 3’ END, WHICH IS TRANSCRIBED LAST, IS
MODIFIED BY ENZYMATIC ADDITION OF A POLYA TAIL, BEFORE THE mRNA EXITS THE NUCLEUS
• POLY(A) TAIL = SEQUENCE OF ABOUT 30 TO 200
ADENINE NUCLEOTIDES ADDED TO THE 3’ END
OF mRNA
– MAY INHIBIT DEGRADATION OF mRNA IN THE
CYTOPLASM
– MAY FACILITATE ATTACHMENT TO SMALL
RIBOSOMAL SUBUNIT
– MAY REGULATE PROTEIN SYNTEHSIS BY
FACILITATING mRNA’S EXPORT FROM NUCLEUS
– IS NOT DIRECTLY ATTACHED TO STOP CODON, BUT TO
TRAILER SEGMENT
• TRAILER SEQUENCE = NONCODING
(UNTRANSLATED) SEQUENCE OF
mRNA FROM THE STOP CODON TO
THE POLY (A) TAIL
RNA PROCESSING VIDEO
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RNA SPLICING
• GENES THAT CODE FOR PROTEINS IN
EUKARYOTES MAY NOT BE
CONTINUOUS SEQUENCES
• INTRONS = NONCODING SEQUENCES IN
DNA THAT INTERVENE BETWEEN
CODING SEQUENCES; ARE
TRANSCRIBED BUT NOT TRANSLATED
• EXONS = CODING SEQUENCES OF A
GENE THAT ARE TRANSCRIBED AND
EXPRESSED
RNA SPLICING
• A PROCESS THAT REMOVES INTRONS
AND JOINS EXONS IN PRE-mRNA;
PRODUCES MATURE mRNA
• snRNPS = SMALL NUCLEAR
RIBONUCLEOPROTEINS; ARE INVOLVED IN
mRNA SPLICING
• SPLICEOSOME = A LARGE MOLECULAR
COMPLEX THAT CATALYZES RNA
SPLICING REACTIONS; MADE OF snRNPS
AND OTHER PROTEINS
SPLICEOSOMES
• AS THE SPICEOSOME IS ASSEMBLED, ONE
TYPE OF snRNP BASE PAIRS WITH A
COMPLEMENTARY SEQUENCE AT THE 5’
END OF THE INTRON
• THE SPLICEOSOME PRECISELY CUTS THE
RNA TRANSCRIPT AT SPECIFIC SITES AT
EITHER END OF THE INTRON, WHICH IS
EXCISED
• THE INTRON IS RELEASED AND THE
ADJACENT EXONS ARE IMMEDIATELY
SPLICED TOGETHER BY THE SPLICEOSOME
RNA SPLICING
ROLES OF snRNPs AND SPICEOSOMES IN mRNA SPLICING
RIBOZYMES
• OTHER KINDS OF RNA PRIMARY
TRANSCRIPTS, SUCH AS THOSE GIVING
RISE TO tRNA AND rRNA, ARE SPLICED BY
MECHANISMS THAT DO NOT INVOLVE
SPLICEOSOMES; HOWEVER, AS WITH
mRNA SPLICING, RNA IS OFTEN INVOLVED
IN CATALYZING THE REACTIONS
• RIBOZYMES = RNA MOLECULES THAT
CAN CATALYZE REACTIONS BY
BREAKING AND FORMING COVALENT
BONDS
PROTEIN SYNTHESIS
• IN A NUTSHELL, PROTEIN SYNTHESIS
IS TAKING THE AMINO ACIDS FROM
FOOD WE EAT AND LINKING THEM
BACK INTO POLYPEPTIDES,
PROTEINS THAT THE BODY USES FOR
VARIOUS FUNCTIONS THROUGHOUT
THE BODY
VIDEO: PROTEIN SYNTHESIS OVERVIEW
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TRANSLATION
TRANSFER RNA (tRNA)
• ALL TYPES OF RNA, INCLUDING tRNA,
ARE TRANSCRIBED FROM TEMPLATE DNA
• tRNA MUST TRAVEL FROM THE NUCLEUS
TO THE CYTOPLASM FOR TRANSLATION
• tRNA MOLECULES CAN BE REUSED
tRNA STRUCTURE
• tRNA IS A SINGLE-STRANDED RNA ONLY ABOUT 80
NUCLEOTIDES LONG
• THE STRAND IS FOLDED, FORMING SEVERAL
DOUBLE-STRANDED REGIONS WHERE SHORT
BASE SEQUENCES OF HYDROGEN BOND WITH
COMPLEMENTARY BASES
• A SINGLE-PLACE VIEW SHOWS A CLOVER LEAF
SHAPE
• A LOOP PROTRUDES A ONE END OF THE L AND HAS
A SPECIALIZED SEQUENCE OF 3 BASES CALLED
THE ANTICODON
• AT THE OTHER END OF THE L PROTRUDES THE 3’
END OF THE MOLECULE, THE ATTACHMENT SITE
FOR AN AMINO ACID
STRUCTURE OF tRNA
WOBBLE EFFECT
• THERE ARE ONLY ABOUT 45 TYPES OF tRNA, BUT
THIS ENOUGH TO TRANSLATE THE 64 CODONS
DUE TO THE WOBBLE EFFECT
• WOBBLE EFFECT = THE ABILITYOF ONE tRNA TO
RECOGNIZE 2 OR 3 DIFFERENT mRNA CODONS;
THE THIRD BASE (5’ END) OF THE tRNA
ANTICODON HAS SOME PLAY OR WOBBLE, SO
THAT IT CAN HYDROGEN BOND WITH MORE THAN
ONE KIND OF BASE IN THE 3RD POSITION (3’END)
OF THE CODON
• A SINGLE tRNA WITH THE ANTICODON CCG WILL
RECOGNIZE THREE mRNA CODONS: GGU, GGC,
GGA, ALL OF WHICH CODE FOR GLYCINE
AMINOACYL-tRNA
SYNTHETASES
• A TYPE OF ENZYME THAT CATALYZES THE
ATTACHMENT OF AN AMINO ACID TO ITS tRNA
• EACH OF THE 20 A.A. HAS A SPECIFIC AMINOACYLtRNA SYNTHETASE
• IN AN ENDERGONIC REACTION DRIVEN BY THE
HYDROLYSIS OF ATP, A SYNTHETASE ATTACHES
AN A.A. TO ITS tRNA IN 2 STEPS:
– 1)ACTIVATION OF THE A.A. WITH AMP
– 2) ATTACHMENT OF THE A.A. TO THE tRNA
AN AMINOACYL-tRNA AT WORK
THE ACTIVE SITE
BIND THE A.A AND
ATP; THE ATP LOSES
2 PHOSPHATES AND
ATTACHES TO THE
A.A. AS AMP
THE tRNA
COVALENTLY
BONDS TO
THE A.A,
DISPLACING
AMP
RIBOSOMES
• RIBOSOMES COORDINATE THE PAIRING
OF tRNA ANTIOCODNS TO mRNA CODONS
• RIBOSOMES HAVE 2 SUBUNITS (SMALL
AND LARGE) WHICH ARE SEPARATED
WHEN NOT INVOLVED IN PROT.
SYNTHESIS
• THEY ARE COMPOSED OF ABOUT 60% RNA
(rRNA) AND 40% PROTEIN
RIBOSOMAL SUBUNITS
• THE SMALL AND LARGE SUBUNITS
ARE:
– CONSTRUCTED IN THE NUCLEOLUS
– DISPATCHED THROUGH NUCLEAR
PORES TO THE CYTOPLASM
– ONCE IN CYTOPLASM, ARE ASSEMBLED
INTO FUNCTIONAL RIBOSOMES ONLY
WHEN ATTACHED TO AN mRNA
RIBOSOMAL BINDING SITES
• 1) mRNA BINDING SITE-CREATES COMPLEX
• 2) P SITE - HOLDS THE tRNA CARRYING THE
GROWING POLYPEPTIDE CHAIN
• 3) A SITE - HOLD THE tRNA CARRYING THE
NEXT A.A. TO BE ADDED
• 4) E SITE - DISCHARGED tRNAs LEAVE THE
RIBOSOME
• ** AS THE RIBOSOME HOLDS THE tRNA AND
mRNA MOLECULES TOGETHER, ENZYMES
TRANSFER THE NEW A.A. FROM ITS tRNA TO
THE CARBOXYL END OF THE GROWING
POLYPEPTIDE
ANATOMY OF A RIBOSOME
BUILDING A POLYPEPTIDE
• THE BUILDING OF A PROTEIN, OR
TRANSLATION, OCCURS IN 3
STAGES:
– 1)INITIATION
– 2) ELONGATION
– 3) TERMINATION
ALL 3 STAGES REQUIRE ENZYMES
AND OTHER PROTEIN FACTORS
INITIATION AND ELONGATION
REQUIRE ENERGY PROVIDED BY
GTP
INITIATION
• INITATION BRINGS TOGETHER mRNA, A tRNA
ATTACHED TO THE FIRST A.A.(METHIONINE),
AND THE 2 RIBOSOMAL SUBUNITS
• A) A SMALL RBIOSOMAL SUBUNIT BINDS TO A
MOLECULE OF mRNA. AN INITIATOR tRNA,
WITH ANTICODON UAC, BASE-PAIRS WITH
START CODON AUG. THIS tRNA CARRIES
METHIONINE
• B) LARGE RIBOSOMAL SUBUNIT ARRIVES. THE
INITIATOR tRNA IS IN THE P SITE. THE A SITE IS
AVAILABLE FOR NEXT A.A.
• GTP PROVIDES THE ENERGY FOR INITIATION
INITIATION OF TRANSLATION
ELONGATION
• SEVERAL PROTEINS CALLED
ELONGATION FACTORS TAKE PART IN
THIS 3-STEP CYCLE, ADDING A.A’S
• 1) CODON RECOGNITION: AN
INCOMING AMINOACYL tRNA BINDS TO
THE CODON IN THE A SITE
– AN ELONGATION FACTOR DIRECTS tRNA
INTO A SITE
– HYDROLYSIS OF GTP PROVIDES ENERGY
• 2) PEPTIDE BOND FORMATION: THE
RIBOSOME CATALYZES THE FORMATION OF A
PEPTIDE BOND BETWEEN THE NEW A.A. AND
THE CARBOXYL END OF GROWING PROTEIN
• 3) TRANSLOCATION- THE tRNA IN THE A SITE,
WHICH IS NOT ATTACHED TO THE GROWING
PEPTIDE, IS MOVED TO P SITE. THE tRNA THAT
WAS IN THE P SITE IS TRANSLOCATED TO THE
E SITE AND EXITS
– THE mRNA IS MOVED THRU RIBOSOME IN THE 5’ TO
3’ DIRECTION
– GTP HYDROLYSIS PROVIDES ENERGY
ELONGATION
TERMINATION
• 1) WHEN RIBOSOME REACHES A
TERMINATION CODON ON mRNA, THE A
SITE ACCEPTS A PROTEIN CALLED A
RELEASE FACTOR
• 2) THE RELEASE FACTOR HYDROLYZES
THE BOND BETWEEN THE tRNA IN THE P
SITE AND THE LAST A.A. OF THE PEPTIDE
CHAIN. IT IS FREED FROM THE
RIBOSOME
• 3) THE TWO RIBOSOMAL SUBUNITS AND
THE OTHER COMPONENTS DISSOCIATE
TERMINATION
TRANSLATION VIDEO
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SUMMARY OF PROTEIN SYNTHESIS
POLYRIBOSOMES
• A SINGLE RIBOSOME CAN MAKE AN AVERAGE
SIZED PROTEIN IN LESS THAN A MINUTE, BUT
CLUSTERS OF RIBOSOMES, CALLED
POLYRIBOSOMES, CAN SIMULTANEOULSY
TRANSLATE AN mRNA MOLECULE
– ONCE A RIBOSOME PASSES THE INITIATION
CODON, A SECOND RIBOSOME CAN ATTACH
TO THE LEADER SEQUENCE OF mRNA
– SEVERAL RIBOSOMES MAY TRANSLATE AN
mRNA AT ONCE, MAKING MANY COPIES OF A
POLYPEPTIDE
POLYRIBOSOMES
FROM POLYPEPTIDE TO
FUNCTIONAL PROTEIN
• THE BIOLOGICAL ACTIVITY OF
PROTEINS DEPENDS UPON A PRECISE
FOLDING OF THE POLYPEPTIDE
CHAIN INTO A 3’D CONFORMATION
– GENES DETERMINE PRIMARY STRUCTURE,
THE LINEAR SEQUENCE OF AMINO ACIDS
– PRIMARY STRUCTURE DETERMINES HOW
THE POLYPEPTIDE CHAIN WILL
SPONTANEOUSLY COIL AND FOLD TO FORM
3-D CONFORMATION
POINT MUTATIONS
• MUTATION = A CHANGE IN THE
GENETIC MATERIAL OF A CELL
• POINT MUTATION = A MUTATION
LIMITED TO ABOUT ONE OR A FEW BASE
PAIRS IN A SINGLE GENE.
• THERE ARE 2 CATEGORIES OF POINT
MUTATIONS:
– 1) BASE-PAIR SUBSTITUTIONS
– 2) BASE-PAIR INSERTIONS OR DELETIONS
BASE-PAIR SUBSTITUTIONS
• THIS IS THE REPLACEMENT OF ONE
BASE PAIR WITH ANOTHER; OCCURS
WHEN A NUCLEOTIDE AND ITS PARTNER
IN THE COMPLEMENTARY DNA STRAND
ARE REPLACED WITH ANOTHER PAIR OF
NUCLEOTIDES
– SOMETIMES HAS LITTLE IF ANY EFFECT
– IT CAN SIGNIFICANTLY ALTER PROTEIN
ACTIVITY
– ON RARE OCCASIONS, CAN IMPROVE PROT.
POINT MUTATIONS
SICKLE CELL-CAUSED BY CHANGE IN BASE PAIR
INSERTIONS OR DELETIONS
• INSERTION - OF ONE OR MORE
NUCLEOTIDE PAIRS INTO A GENE
• DELETION - OF ONE OR MORE
NUCLEOTIDE PAIRS INTO A GENE
• MAY ALTER THE READING FRAME
(TRIPLET GROUPING). THIS TYPE OF
FRAMESHIFT MUTATION WILL OCCUR
WHEN NUMBER OF NUCLEOTIDES
INSERTED OR DELETED IS NOT A
MULTIPLE OF 3
• THIS WILL PRODUCE A NONFUNCTIONAL
PROTEIN UNLESS IT IS VERY NEAR END
OF GENE
MUTAGENS
• MUTAGENESIS = THE CREATION OF
MUTAGENS, WHICH CAN OCCUR AS
ERRORS IN DNA REPLICATION, REPAIR,
OR RECOMBINATIONS THAT RESULT IN
BASE-PAIR SUBSTITUTIONS, INSERTIONS
OR DELETION
• MUTAGEN = PHYSICAL OR CHEMICAL
AGENTS THAT INTERACT WITH GENETIC
MATERIAL TO CAUSE MUTATIONS
– RADIATION MOST COMMON PHYSICAL
MUTATION