central dogma review of dna clip 2 dna replication review clip 1
TRANSCRIPT
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Central Dogma
Review of DNA Clip 2
DNA replication review Clip 1
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Some BIG IDEAS
The Connection Between Genes & Proteins
• Transcription and translation are the two Transcription and translation are the two
main processing linking gene to proteinmain processing linking gene to protein• Many enzymes are involved in this processMany enzymes are involved in this process• In the genetic code, nucleotide triplets In the genetic code, nucleotide triplets
specify amino acidsspecify amino acids
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• The info contained in DNA is in the form of specific sequences of nucleotides along the DNA strands.
• The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins.
• Proteins are the links between genotype and phenotype.
Introduction
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• Genes (Segments of DNA) provide the instructions for making specific proteins.
• The bridge between DNA and protein synthesis is RNA.
Reminders:Reminders:
• RNA is chemically similar to DNA, except that it contains ribose as its sugar and substitutes the nitrogenous base uracil for thymine.
• Single stranded.
TranscriptionTranscription and translation are the two main processes in protein
synthesis
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Transcription: The PlayersTranscription: The Players DNADNA mRNAmRNA EnzymesEnzymes
RNA PolymeraseRNA Polymerase Other ‘players’:Other ‘players’: RNA nucleotidesRNA nucleotides
transcription factors (proteins)transcription factors (proteins) TATA BoxTATA Box promoters/terminator promoters/terminator
sequences sequences
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Step 1: Step 1: TranscriptioTranscriptio
nn
Overview:Overview:• During transcription, a DNA strand provides a template for DNA strand provides a template for
the synthesis of a complementary RNA strandthe synthesis of a complementary RNA strand.
• Transcription of a gene produces a messenger RNA (mRNA) molecule.
• In a eukaryotic cell, almost all transcription occurs in the nucleus
Adenine (DNA and RNA)Cystosine (DNA and RNA)Guanine(DNA and RNA)Thymine (DNA only)Uracil (RNA only)
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3 stages of 3 stages of
Transcription:Transcription:
1. Initiation
2. Elongation
3. Termination
Overview
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Some players:• Promoter: sequence in DNA where RNA
polymerase attaches and initiates transcription
• Terminator: sequence that signals the end of transcription
• Transcription unit: stretch of DNA that is being copied into mRNA
Transcription Overview Animation
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1. RNA polymerase attaches and initiates transcription at the Promoter site
1) Initiation (basic)
Animation
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• RNA polymerasebinds to transcriptionfactors to create atranscriptioninitiation complex
• (large complex of about 50 different proteins).
• RNA polymerase IIthen startstranscription (5’ to 3’ direction)
1)Initiation(more detailed)
•Transcription factors include a wide number of proteins that initiate and regulate the transcription of genes.•They have the ability to bind to specific sequences of DNA called enhancer or promoter sequences
transcriptionfactors Clip
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•In prokaryotes, RNA polymerase can recognize and bind directly to the Promoter region
•In eukaryotes, the transcription factors recognize the Promoter region TATAAAA… (usually called a TATA box) AND THEN RNA poly binds.
1) Initiation
Animation 2
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A little more on RNA Polymerase:
• Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules.
• In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their nuclei.• RNA polymerase II is used for mRNA synthesis.
• RNA polymerase I & III transcribes the genes for ribosomal RNA
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• ▪As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time.
• RNA polymerases can add nucleotides only to the 3’ end of the growing polymer.
• Behind the pointof RNA synthesis,the double helixre-forms and theRNA moleculepeels away.
Elongation
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• DNA strand- provides a template for ordering the sequence of nucleotides in an RNA transcript.
• The complementary RNA molecule is synthesized according to base-pairing rules, except that uraciluracil is the complementary base to adenine.
Elongation
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• A single gene can be transcribed simultaneously by several RNA polymerases at a time.
• A growing strand of RNA trails off from each polymerase.• The length of each new strand reflects how far
along the template the enzyme has traveled from the start point.
• The congregation of many polymerase molecules simultaneously transcribing a single gene increases the amount of mRNA transcribed from it.
• This helps the cell make the encoded protein in large amounts.
Elongation: A little more info
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• ProkaryotesProkaryotes-stops right at the end of the terminator.• Both the RNA and DNA is
then released.
• EukaryotesEukaryotes-continues for hundreds of nucleotides past the terminator sequence, AAUAAA.• At a point about 10 to
35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme.
TerminationTranscription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA.
Animation
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Transcription Review Clip
Transcription website
(on your own)
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• Before the primary transcript can leave the nucleus it is modified in various ways during RNA processing before the finished
mRNA is exported to the cytoplasm.
mRNA Processing
1.5’ cap2.poly(A) tail3.RNA splicing:
Introns and exons
eukaryotes
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• At the 5’ end of the pre-mRNA molecule, a a modified form of guanine is addedmodified form of guanine is added, the 5’ cap.
• This helps protect mRNA from hydrolytic enzymes.
• It also functions as an “attach here” signalsignal for ribosomes.
5’ cap
mRNA Processing
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• At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly(A) tail.• In addition to inhibiting hydrolysis and facilitating
ribosome termination, the poly(A) tail also seems to facilitate the export of mRNA from the nucleus.
• The mRNA molecule also includes nontranslated leader and trailer segments.
poly(A) tail
mRNA Processing
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• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides.
• Noncoding segments, introns.
• The final mRNA transcript includes coding regions, exons, plus the leader and trailer sequences.
RNA splicing
mRNA Processing
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Fig. 17.9
RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.
RNA splicing
mRNA Processing
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• This splicing is accomplished by a spliceosome.• spliceosomes
consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs).
• Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA).
RNA splicing Splicing Clip
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• In this process, the snRNA acts as a ribozyme, an RNA molecule that functions as an enzyme.
• The discovery of ribozymes rendered obsolete the statement, “All biological catalysts are proteins.”
RNA splicing
Pro vs, Eu mRNA
processing REVIEW CLIP
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RNA splicing appears to have several RNA splicing appears to have several functions. functions.
1. First, at least some introns contain sequences that control gene activity in some way.
2. Splicing itself may regulate the passage of mRNA from the nucleus to the cytoplasm.
3. One clear benefit of split genes is to enable a one gene to encode for more than one polypeptide.
• Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons.
• Early results of the Human Genome Project indicate that this phenomenon may be common in humans.
RNA splicing
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TRANSLATION(protein is made on ribosome)
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TranslationDuring
translation, the information contained in the order of nucleotides in mRNA is used to determine the amino acid sequence of a polypeptide.
Translation occurs at ribosomes. Translation occurs mainly at ribosomes in the cytoplasm
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• Ribosomes:
• Remember:
• There are 2 populations of ribosomes, free and bound, are active participants in protein synthesis.
• Free ribosomes are suspended in the cytosol and synthesize proteins that reside in the cytosol.
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While very similar in structure and function, prokaryotic and eukaryotic ribosomes have enough differences that certain antibiotic drugs (like tetracycline) can paralyze prokaryotic ribosomes without inhibiting eukaryotic ribosomes.
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Ribosomes attached to the ER
Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum.
They synthesize proteins of the endomembrane system as well as proteins secreted from the cell
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• AND AND RemembeRemember…r…Proteins are made out of amino acids.
• There are 20 different amino acids.
• Side chains determine properties
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Translation: The PlayersTranslation: The Players
1.1. tRNAtRNA
2.2. mRNAmRNA
3.3. rRNArRNA
4.4. RibosomesRibosomes
5.5. Amino acidsAmino acids
6.6. EnzymesEnzymes
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• Transfer RNATransfer RNA (tRNA) transfers amino acids from the cytoplasm’s pool to a ribosome.
• RNA (mRNA)RNA (mRNA) brings the code to the ribosome.
• The ribosomeThe ribosome adds each amino acid carried by tRNA to the growing end of the polypeptide chain.
Translation
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Transfer Transfer RNARNA (tRNA)
• Each tRNA arriving at the ribosome carries a specific amino acid at one end and has a specific nucleotide triplet, an anticodonanticodon, at the other.
• The anticodon base-pairs with a complementary triplet codoncodon on mRNA.
mRNA
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A A ttRNA molecule consists of a strand of RNA molecule consists of a strand of about 80 nucleotides that folds back on about 80 nucleotides that folds back on itself to form a three-dimensional itself to form a three-dimensional structure. structure. (held together by H-bond)(held together by H-bond) It includes a loop containing the anticodon and an It includes a loop containing the anticodon and an
attachment site at the 3’ end for an amino acid.attachment site at the 3’ end for an amino acid.
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• Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase.
• The 20 different synthetases match the 20 different amino acids.
• Each has active sites for only a specific tRNA and amino acid combination.
aminoacyl-tRNA
synthetase CLIP
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Ribosomes facilitate the specific coupling of the tRNA anticodons with mRNA codons.• Each ribosome has a large and a small subunit.
• These are composed of proteins and ribosomal RNA (rRNA), the most abundant RNA in the cell.•The large
and small subunits join to form a functional ribosome only when only when they they attachattach to an mRNA molecule.
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Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.
• The P siteP site holds the tRNA carrying the growing polypeptide chain.
• The A siteA site carries the tRNA with the next amino acid.
• Discharged tRNAs leave the ribosome at the E siteE site.
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Translation can be divided into three stages:
1.initiation
2.elongation
3.termination
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• InitiationInitiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits.1. a small ribosomal subunit binds with mRNA and a special
initiator tRNA, which carries methionine and attaches to the start codon (AUG)
2. Initiation factors bring in the large subunit such that the initiator tRNA occupies the P site.
Energy provided
by….
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• Elongation consists of a series of three-step cycles as each amino acid is added (peptide bond formed) to the proceeding one.
Clip
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•An elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresonding anticodon of tRNA carrying the appropriate amino acid.This step requires the hydrolysis of two GTP.
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• During peptide bond formation, an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site.
•This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, now one amino acid longer, to the tRNA at the A site.
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• During translocation, the ribosome moves the tRNA with the attached polypeptide from the A site to the P site.
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• The three steps of elongation continue codon by codon to add amino acids until the polypeptide chain is completed.
• STOP codon is reached .
How does it know which amino acids are
correct
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mRNA Start codon
Ribosome
Methionine
Phenylalanine tRNALysine
Nucleus
Translation
mRNA
Go to Section:
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Mid-1960s the entire Mid-1960s the entire code was deciphered.code was deciphered.
The codon AUG not The codon AUG not only codes for the only codes for the amino acid methionine amino acid methionine but also indicates the but also indicates the start of translationstart of translation..
ThreeThree codons do codons do not indicate amino not indicate amino acids but signal acids but signal the termination the termination of translation.of translation.
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•The anticodons of some tRNAs recognize more than one codon.
•Wobble
•1966 Francis Crick proposed the Wobble hypothesis
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mRNARibosome
Translation direction
LysinetRNA
tRNA
Ribosome
Growing polypeptide chain
mRNA
Section 12-3
Go to Section:
Translation
•Codon by codon, tRNAs deposit amino acids in the prescribed order and the ribosome joins them into a polypeptide chain.
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• TerminationTermination occurs when one of the three stop codons reaches the A site.
• A release factorrelease factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site.
• This frees the polypeptide and the translation complex disassemblesdisassembles.
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• Typically a single mRNAa single mRNA is used to make many copies of a polypeptide simultaneously.
• Multiple ribosomesMultiple ribosomes, polyribosomes, may trail along the same mRNA
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Translation Review Clip
Translation website
Molecular Visions
Clip
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• Although bacteria and eukaryotes carry out transcription and translation in very similar ways, they do have differences in cellular machinery and in details of the processes.• Eukaryotic RNA polymerases require
transcription factors.
• Ribosomes are also different.
• Prokaryotic- Transcription & Translation can happen at the same time
• Eukaryotes- mRNA processing
Comparing protein synthesis in prokaryotes and eukaryotes: a
review
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• The basic mechanics of transcription and translation are similar in eukaryotes and prokaryotes.
• Because bacteria lack nuclei, transcription and translation are coupled.
Bacterial Protein Synthesis
•Ribosomes attach to the leading end of a mRNA molecule while transcription is still in progress.
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• One big difference is that prokaryotes can transcribe and translate the same gene simultaneously
• The new protein quickly diffuses to its operating site.
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• In eukaryotes, extensive RNA processing is inserted between transcription and translation.
• In addition, eukaryotic cells have complicated mechanisms for targeting proteins to the appropriate organelle. (free and bound ribosomes)
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• The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals.
• In laboratory experiments, genes can be transcribed and translated after they are transplanted from one species to another.
• This tobacco plant is expressinga transpired firefly gene.
The genetic code must have evolved very early in the
history of life
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• This has permitted bacteria to be programmed to synthesize certain human proteins after insertion of the appropriate human genes.-insulin
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The near universality of the genetic code must have been
operating very early in the history of life.
A shared genetic vocabulary is a reminder of the kinship that
bonds all life on Earth.
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Gene Regulation (what gets made & when…)
• Eukaryotes• Enhancers can bind to sequences upstream. Then
bind to the TFs in the promoter site. (TFs can be thought if as activators)
• Silences (same as above, just turn gene off)• Methylation of DNA• mRNA processing before it leaves the nucleus
• Gene regulation in Bacteria: • The Operon- A group, or cluster, of genes (that make
work together) or a segment of DNA that functions as a single transcription unit. It is comprised of an operator, a promoter, and one or more structural genes that are transcribed into one mRNA
• Inducible or repressible• Example Lac Operon (inducible) Try Operon
(repressible)
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Mutations
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Mutations in DNA usually occur through one of two
processes:1- DNA damage from
environmental agents such as ultraviolet light, nuclear radiation or certain chemicals.
2- Errors that occur when a cell replicates its DNA in preparation for cell division.
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Mutations are changes in the genetic material of a cell (or virus).
• These include large-scale mutations in which long segments of DNA are affected
• translocations, duplications, inversions.
• A chemical change in just one base pair of a gene causes a point mutation.
• deletions, insertions, substitutions
• These mutations will only be transmitted to future generations if they occur in gametes or cells producing gametes.
DNA Poly proofreading function Clip
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point mutations
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Substitutions (3 outcomes)
*no change (silent mutations) *missense mutations *nonsense mutations
Point Mutations
-most common-Substitution, deletions,
insertions
Missense mutations are those that still code for an amino acid but change the indicated amino acid.Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.
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Sickle-cell anemia is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin.
• A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.
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Insertions and
deletions
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• Insertions and deletions are additions or losses of nucleotide pairs in a gene.• These have a disastrous
effect on the resulting protein more often than substitutions do.
• Unless these mutations occur in multiples of three, they cause a frameshift mutationframeshift mutation.• All the nucleotides
downstream of the deletion or insertion will be improperly grouped into codons.
• The result will be extensive missense, ending sooner or later in nonsense - premature termination.
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Deletion
InsertionClip
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Substitution Insertion Deletion
Substitution, Insertion, & Deletion
Go to Section:
Gene Mutations only affect ONE point of the code -- often called
Point Mutations…
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ChromosomalMutations
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Duplication
Inversion
Translocation
Chromosomal Mutations – affects LARGE portions of the code (entire genes
or entire chromosomes…)
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Inversions•In an inversion mutation, an entire section of DNA is reversed. A small inversion may involve only a few bases within a gene, while longer inversions involve large regions of a chromosome containing several genes.•The new sequence may not be viable to produce an organism, depending on which genes are reversed. Advantageous characteristics from this mutation are also possible
Original The fat cat ate the wee rat.
Insertion The fat tar eew eht eta tac.
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•Genetic mutations increase genetic diversity and therefore have an important role in evolution.
• They are also the reason many people inherit diseases.
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The Mendelian concept of a gene views it as a discrete unit of inheritance that affects phenotype.
Morgan and his colleagues assigned genes to specific loci on chromosomes.
We can also view a gene as a specific nucleotide sequence along a region of a DNA molecule.
We can define a gene functionally as a DNA sequence that codes for a specific polypeptide chain.
What is a gene?
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