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Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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Page 1: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Central Dogma

Review of DNA Clip 2

DNA replication review Clip 1

Page 2: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 3: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 4: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 5: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 6: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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)

Page 7: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

3 stages of 3 stages of

Transcription:Transcription:

1. Initiation

2. Elongation

3. Termination

Overview

Page 8: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 9: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

1. RNA polymerase attaches and initiates transcription at the Promoter site

1) Initiation (basic)

Animation

Page 10: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 11: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

•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

Page 12: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 13: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• ▪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

Page 14: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 15: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 16: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 18: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 19: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 20: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 21: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 22: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Fig. 17.9

RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.

RNA splicing

mRNA Processing

Page 23: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 24: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 25: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 26: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

TRANSLATION(protein is made on ribosome)

Page 27: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 28: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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.

Page 29: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 30: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 31: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• AND AND RemembeRemember…r…Proteins are made out of amino acids.

• There are 20 different amino acids.

• Side chains determine properties

Page 32: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 33: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 34: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 35: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 36: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 37: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 38: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 39: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Translation can be divided into three stages:

1.initiation

2.elongation

3.termination

Page 40: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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….

Page 41: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• Elongation consists of a series of three-step cycles as each amino acid is added (peptide bond formed) to the proceeding one.

Clip

Page 42: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

•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.

Page 43: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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.

Page 44: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• During translocation, the ribosome moves the tRNA with the attached polypeptide from the A site to the P site.

Page 45: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 46: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

mRNA Start codon

Ribosome

Methionine

Phenylalanine tRNALysine

Nucleus

Translation

mRNA

Go to Section:

Page 47: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 48: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1
Page 49: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

•The anticodons of some tRNAs recognize more than one codon.

•Wobble

•1966 Francis Crick proposed the Wobble hypothesis

Page 50: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 51: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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.

Page 52: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 54: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 55: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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.

Page 56: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• One big difference is that prokaryotes can transcribe and translate the same gene simultaneously

• The new protein quickly diffuses to its operating site.

Page 57: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 59: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• This has permitted bacteria to be programmed to synthesize certain human proteins after insertion of the appropriate human genes.-insulin

Page 60: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 61: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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)

Page 62: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Mutations

Page 63: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Page 64: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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

Page 65: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

point mutations

Page 66: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

Clip

Page 67: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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.

clip

Page 68: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Insertions and

deletions

Page 69: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

• 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

Page 71: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Substitution Insertion Deletion

Substitution, Insertion, & Deletion

Go to Section:

Gene Mutations only affect ONE point of the code -- often called

Point Mutations…

Page 72: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

ChromosomalMutations

Page 73: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

Duplication

Inversion

Translocation

Chromosomal Mutations – affects LARGE portions of the code (entire genes

or entire chromosomes…)

Page 74: Central Dogma Review of DNA Clip 2 DNA replication review Clip 1

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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