DNA Structure and Function

DNA Structure and Function
Replication, Transcription and Translation, Chomosome Structure and Mutation Review

DNA Structure

DNA DNA is often called the blueprint of life
In simple terms, DNA contains the instructions for making proteins within the cell

DNA Late 1800’s – Biochemists know nucleic acids are composed of sugars, phosphoric acid, and several nitrogen containing bases

DNA By the early 1900’s it was known that the chromosomes carry the genetic (hereditary) information Chromosomes consist of DNA (deoxyribonucleic acid) and proteins Which one of these chemical components carries the information?

Key Experiments 1928 Frederick Griffith Transformation Exp.
1944 Oswald Avery, Maclyn McCarty, & Colin MacLeod Isolate Transforming Agent 1952 Alfred Hershey & Martha Chase

Frederick Griffith Discovers that a factor in diseased bacteria can transform harmless bacteria into deadly bacteria (1928)

Key Experiments 1928 Frederick Griffith Transformation Exp.
1944 Oswald Avery, Maclyn McCarty, & Colin MacLeod Isolate Transforming Agent 1952 Alfred Hershey & Martha Chase

DNA & Proteins 1948 – Linus Pauling discovers many proteins have an alpha helical structure

Important Discoveries in DNA Structure
Erwin Chargaff Discovered the relationships between DNA bases Maurice Wilkins, Rosalind Franklin and colleagues Discovered the basic structure of DNA by x-ray crystallography James Watson and Francis Crick Built the first accurate model of a DNA molecule

Key Players Erwin Chargaff, Maurice Wilkins, Rosalind Franklin, James Watson, and Francis Crick

Four nitrogenous bases
DNA has four different bases: Cytosine C Thymine T Adenine A Guanine G

Chargaff’s Rules Chargaff’s data showed that in each species DNA, the percent of A equals the percent of T, and the percent of G equals the percent of C Proportions of A/T and G/C vary among diferent species

Chargaff’s Rule: Adenine and Thymine always join together A T
Cytosine and Guanine always join together C G

One deoxyribose together with its phosphate and base make a nucleotide
Nucleotides O -P O O One deoxyribose together with its phosphate and base make a nucleotide O -P O O Nitrogenous base C O Phosphate C C C Deoxyribose ribose O

RNA vs. DNA DNA Double stranded Deoxyribose sugar Bases: C,G A,T RNA
Single stranded Ribose sugar Bases: C,G,A,U Both contain a sugar, phosphate, and base.

Nucleic Acids DNA is the genetic material
made of 4 building blocks – nucleotides adenine (A), guanine (G), cytosine (C), thymine (T) A-T, C-G in the chain double helix model double stranded DNA RNA carries hereditary information from nuclear DNA to the cytoplasm (inside cells) uracil (U) replaces T single stranded RNA Complimentary Base-Pairing

Two Kinds of Bases in DNA
Pyrimidines are single ring bases. Purines are double ring bases. N C O C C N C C N

Thymine and Cytosine are Pyrimidines
Thymine and cytosine each have one ring of carbon and nitrogen atoms. C N O cytosine C N O thymine

Adenine and Guanine are Purines
Adenine and guanine each have two rings of carbon and nitrogen atoms. C N O Guanine C N Adenine

James Watson & Francis Crick:
Used data of M.H.F. Wilkins and Rosalind Franklin, early 50’s Wilkins and Franklin studied the structure of DNA crystals using X-rays The X pattern suggested the structure of DNA was a helix Distance between the two “backbones” of DNA is constant along the length of the molecule

One Strand of DNA phosphate The backbone of the molecule is alternating phosphates and deoxyribose sugars The stair steps are nitrogenous bases DNA is a polymer of nucleotides (thousands of repeating units that make up the DNA) deoxyribose bases

Hydrogen Bonds The bases attract each other because of hydrogen bonds.
Hydrogen bonds are weak but there are millions and millions of them in a single molecule of DNA. The bonds between cytosine and guanine are shown here with dotted lines

Base Pairing Rule C N O When making hydrogen bonds, cytosine always pairs up with guanine Adenine always pairs up with thymine Adenine is bonded to thymine here

Base Pairing Rule

The Double Helix A DNA molecule consists of two strands of nucleotide monomers running in opposite directions in a ladder-like structure which is twisted or coiled into a double helix

The Double Helix

The Structure of the Genetic Material

DNA Structure Watson, J.D. & F.H.C. Crick Molecular Structure of Nucleic Acid: A Structure for Deoxyribonucleic Acid. Nature 171:737. Wilkins, M. Stokes A.F., Wilson H.R Molecular Structure of Deoxypentose Nucleic Acids. Nature 171: 738. Franklin, R. & Gosling R Molecular Configuration in Sodium Thymonucleate. Nature 171: 740. Franklin, R Evidence for 2-Chain Helix in Crystalline Structure of Sodium Deoxyribonucleate. Nature. 174: 156. Watson, Crick & Wilkins receive Nobel Prize in 1962 for deciphering structure of DNA

DNA Fun Facts Each cell has about 2 m of DNA
The average human has 75 trillion cells The average human has enough DNA to go from the earth to the sun more than 400 times. DNA has a diameter of only m The earth is 150 billion m or 93 million miles from the sun If you unravel all the DNA in the chromosomes of one of your cells, it would stretch out 2 meters. If you did this to the DNA in all your cells, it would stretch from here to sun more than 400 hundred times!

Watson and Crick’s Double Helix Model explained:
1. How replication of DNA during mitosis produces exact copies for the daughter cells 2. How DNA acts as a code, specifying how proteins are made by the cell

The Basic Principle: Base Pairing to a Template Strand
The relationship between structure and function is manifest in the double helix Since the two strands of DNA are complementary each strand acts as a template for building a new strand in replication

How Does DNA Act As A Code?
The order of bases on the DNA strand instructs the ribosomes how to synthesize proteins

Information Transfer — DNA to RNA to Protein
DNA triplets transcribed to produce complementary codon triplets in mRNA Each mRNA codon triplet specifies a particular amino acid Sequentially, codon by codon, the mRNA is a blueprint used in translation to produce a particular protein molecule

If the order of bases along one strand of DNA is AGGTTACTGCAC what is the order of bases on the complementary strand? TCCAATGACGTG

Patterns of Base Pairing
The order of bases (DNA sequence) varies among species and among individuals Each species has characteristic DNA sequences DNA sequence The order of nucleotide bases in a strand of DNA

Gene: the portion of a DNA molecule that codes for the production of a specific polypeptide or protein

DNA Replication

The Cell Cycle Duplicated chromosomes are composed of 2 DNA molecules

The Cell Cycle Duplicated chromosomes are composed of 2 DNA molecules
During S phase each strand of a DNA molecule serves as a template for the formation of a new strand

DNA Replication

DNA Replication in Prokaryotes and Eukaryotes
Overall mechanism Roles of Polymerases & other proteins More mechanism: Initiation and Termination Mitochondrial DNA replication

Replication DNA double helix unwinds DNA now single-stranded
New DNA strand forms using complementary base pairing (A-T, C-G) Used to prepare DNA for cell division Whole genome copied/replicated

DNA Replication semi-conservative helicase – unwinds DNA
DNA polymerases one strand is the template builds a complementary strand bases pair with hydrogen bonds A-T C-G

DNA replication is semi-conservative, i. e
DNA replication is semi-conservative, i.e., each daughter duplex molecule contains one new strand one old.

DNA Replication is Semi-Conservative
Each 2-stranded daughter molecule is only half new One original strand was used as a template to make the new strand

DNA Replication Meselson-Stahl Exp.
Semi-conservative Model of DNA replication

DNA Replication The copying of DNA is remarkable in its speed and accuracy Involves unwinding the double helix and synthesizing two new strands. More than a dozen enzymes and other proteins participate in DNA replication The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated

DNA Replication Origins
Replicon - DNA replicated from a single origin DNA Replication Origins Table in notes Eukaryotes have many replication origins

DNA Replication

1) The two strands of a DNA molecule are complementary: their nucleotides match up according to base-pairing rules (G to C, T to A). 2) As replication starts, the two strands of DNA unwind at many sites along the length of the molecule. 3) Each parent strand serves as a template for assembly of a new DNA strand from nucleotides, according to base-pairing rules. Figure 6.8: Animated! DNA replication. Each strand of a DNA double helix is copied; two double-stranded DNA molecules result. 4) DNA ligase seals any gaps that remain between bases of the “new” DNA, so a continuous strand forms. The base sequence of each half-old, half-new DNA molecule is identical to that of the parent.

DNA Replication

DNA Replication: The Double Helix

DNA Replication Enzymes
DNA Primase, DNA Replicase, DNA Polymerase III, DNA Polymerase I (DNA Polymerse II is a repair enzyme)

Enzymes of DNA Replication
1. DNA-dependent DNA polymerases synthesize DNA from nucleotide triphosphate monomers require a template strand and an RNA primer strand with a 3’-OH end all enzymes synthesize from 5’ to 3’ (add nucleotides to 3’ end only)

If DNA polymerases only synthesize 5’ to 3’, how does the replication fork move directionally?

DNA Replication The Lagging strand is synthesized as small (~10bp) fragments - “Okazaki fragments” Okazaki fragments begin as very short nucleotide RNA primers synthesized by primase 2. Primase - the RNA polymerase that synthesizes the RNA primers (11-12 nucleotides that start with pppAG) for both lagging and leading strand synthesis

Mechanism of DNA Replication
DNA replication is catalyzed by DNA polymerase which needs an RNA primer RNA primase synthesizes primer on DNA strand DNA polymerase adds nucleotides to the 3’ end of the growing strand

Mechanism of DNA Replication
DNA polymerase I degrades the RNA primer and replaces it with DNA

Lagging Strand Synthesis (continued)
Polymerase III extends the RNA primers until the 3’ end of an Okazaki fragment reaches the 5’ end of a downstream Okazaki fragment Then, Polymerase I degrades the RNA part with its 5’- 3’ exonuclease activity, and replaces it with DNA Polymerase I (the first to be discovered) is not highly processive, so it stops before going too far

At this stage, the Lagging strand is a series of DNA fragments (without gaps)
Okazaki fragments are stitched together covalently by DNA Ligase DNA Ligase - joins the 5’ phosphate of one DNA molecule to the 3’ OH of another, using energy in the form of NAD (prokaryotes) or ATP (eukaryotes) DNA Ligase prefers substrates that are double-stranded, with only one strand needing ligation, and lacking gaps

+ + Mechanism of Prokaryotic DNA Ligase
Ligase cleaves NAD and attaches to AMP. Ligase-AMP binds and attaches to 5’ end of DNA #1 via the AMP. The 3’OH of DNA #2 reacts with the phosphodiester shown, displacing the AMP-ligase. AMP & ligase separate. HO P 3' 5' Ligase P NAD NMN +AMP 1 N M N 3' P Ligase 1 + P AMP AMP 3' N A D 2 HO Also in notes + AMP 1 2 (Euk. DNA ligase uses ATP as AMP donor) P 3' 5'

Other proteins needed for DNA replication:
DNA Helicase (dnaB gene) – hexameric protein, unwinds DNA strands, uses ATP SSB – single-strand DNA binding protein, prevents strands from re-annealing and from being degraded, stimulates DNA Polymerase III Gyrase – a.k.a. Topoisomerase II, keeps DNA ahead of fork from over winding (i.e., relieves torsional strain) Replisome - DNA and protein machinery at a replication fork

The Mechanism of DNA Replication
Many proteins assist in DNA replication DNA helicases unwind the double helix, the template strands are stabilized by other proteins Single-stranded DNA binding proteins make the template available RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added. DNA polymerase III extends the strand in the 5’-to-3’ direction DNA polymerase I degrades the RNA primer and replaces it with DNA DNA ligase joins the DNA fragments into a continuous daughter strand

Enzymes in DNA replication
Primase adds short primer to template strand Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase III binds nucleotides to form new strands DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone

DNA Replication Helicase protein binds to DNA sequences called
Primase protein makes a short segment of RNA complementary to the DNA, a primer 5’ 3’ Binding proteins prevent single strands from rewinding 3’ 5’ Helicase protein binds to DNA sequences called origins and unwinds DNA strands

DNA Replication Overall direction of replication 5’ 3’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer

DNA Replication DNA polymerase enzyme adds DNA nucleotides
5’ Overall direction of replication 3’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer DNA polymerase proofreads bases added and replaces incorrect nucleotides

DNA Replication 5’ 3’ Overall direction of replication Leading strand synthesis continues in a 5’ to 3’ direction

DNA Replication Leading strand synthesis continues in a
3’ 5’ Overall direction of replication Okazaki fragment Leading strand synthesis continues in a 5’ to 3’ direction Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments

DNA Replication Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments

DNA Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments

DNA Replication 5’ 3’ 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments

DNA Replication 3’ 5’ 5’ 5’ 3’ Exonuclease activity of DNA polymerase I removes RNA primers

DNA Replication Polymerase activity of DNA polymerase I fills the gaps
3’ 5’ Polymerase activity of DNA polymerase I fills the gaps Ligase forms bonds between sugar-phosphate backbone

DNA Replication

Replication Fork Overview

Origins of Replication
Helicase Replication Forks

Helicase Replication Forks Primase DNA Polymerase

Helicase Replication Forks Primase DNA Polymerase Nuclease Ligase

DNA Replication Helicase Replication Forks Primase DNA Polymerase Nuclease Ligase Telomerase

DNA Replication in a Prokaryote
The circular bacterial DNA begins replication at a single site, the replication origin Replication proceeds out in both directions, until copies of each strand of DNA are produced

Replication Causes DNA to Supercoil

Rubber Band Model of Supercoiling DNA
DNA Gyrase relaxes positive supercoils by breaking and rejoining both DNA strands. Figure not in the 4th Edition.

DNA Replication and Repair
A cell replicates its DNA before it divides Each strand of the double helix serves as a template for synthesis of a new, complementary strand of DNA DNA replication results in two double-stranded DNA molecules identical to the parent

During DNA replication, the double-helix unwinds DNA polymerase uses each strand as a template to assemble new, complementary strands of DNA from free nucleotides DNA ligase seals any gaps to form a continuous strand

Duplication of a cell’s DNA before cell division DNA polymerase DNA replication enzyme; assembles a new strand of DNA based on sequence of a DNA template DNA ligase Enzyme that seals breaks in double-stranded DNA

Accuracy of DNA Replication
The chromosome of E. coli bacteria contains about 5 million bases pairs Capable of copying this DNA in less than an hour The 46 chromosomes of a human cell contain about 6 BILLION base pairs of DNA Printed one letter (A,C,T,G) at a time…would fill up over 900 volumes of a phone book Takes a cell a few hours to copy this DNA With amazing accuracy – an average of 1 error per billion nucleotides

Checking for Mistakes DNA repair mechanisms fix damaged DNA
Proofreading by DNA polymerase corrects most base-pairing errors DNA repair mechanisms Any of several processes by which enzymes repair DNA damage

Proofreading DNA must be faithfully replicated…but mistakes occur
DNA polymerase (DNA pol) inserts the wrong nucleotide base in 1/10,000 bases DNA pol has a proofreading capability and can correct errors Mismatch repair: ‘wrong’ inserted base can be removed Excision repair: DNA may be damaged by chemicals, radiation, etc. Mechanism to cut out and replace with correct bases

Proofreading and Repairing DNA
DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing In nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNA Nuclease DNA polymerase ligase A thymine dimer distorts the DNA molecule. 1 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. 2 Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. 4

Proofreading and Repairing DNA
DNA Repair Mechanisms DNA Polymerase Proofreading ability Mismatch Repair Damage Repair Enzymes involved: Nucleases DNA Repair Polymerase Ligase

Transcription & Translation

In Organisms, Information Flows from DNA to RNA to Proteins
The Central Dogma Of Molecular Biology The Genetic Code Is Universal and Redundant Unambiguous

From Gene to Protein

Flow of Genetic Information in Cells
Prokaryotes Transcription & Translation occur in the general cytoplasm Eukaryotes Processes separated by the nuclear envelope Primary Transcript is modified in the nucleus processing

Protein Synthesis The information content of 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 The process by which DNA directs protein synthesis, gene expression includes two stages, called transcription and translation

Two Stage Protein Synthesis
Transcription (in nucleus*) DNA – gene blue print triplet code - 3 bases/AA exons - expressed introns – excised RNA – tools for protein synthesis mRNA tRNA rRNA Translation (in cytoplasm) Ribosome codons are read to build a primary protein structure * No nucleus in the prokaryotes

Transcription and Translation
Cells are governed by a cellular chain of command DNA RNA protein Transcription Is the synthesis of RNA under the direction of DNA Produces messenger RNA (mRNA) Translation Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA Occurs on ribosomes

DNA  RNA  Protein Transcription –
Translation Transcription – is the formation of an RNA molecule based upon a gene sequence Different types of RNA can be formed depending on the Gene being transcribed. The Main Types are: Messenger RNA, Transfer RNA, Ribosomal RNA.

DNA  RNA  Protein Translation –
Transcription Translation Translation – is the formation of a Protein molecule based upon an mRNA sequence While the mRNA sequence provides the information to form the protein’s sequence, two other types of RNA are needed during translation

Protein Structure Made up of amino acids
Polypeptide – linear string of amino acids joined by peptide bonds 20 amino acids are arranged in different orders to make a variety of proteins Assembled on a ribosome

20 Amino Acids Used in Protein Synthesis

Three RNAs in Protein Synthesis
Different types of RNA play a different role in the synthesis of protein: mRNA rRNA tRNA

Transcription and Translation
In a eukaryotic cell the nuclear envelope separates transcription from translation Extensive RNA processing occurs in the nucleus Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. (b) TRANSCRIPTION RNA PROCESSING TRANSLATION mRNA DNA Pre-mRNA Polypeptide Ribosome Nuclear envelope

Translation Uses the Codon Dictionary
alternate CUG start codon in some vertebrate immune cells The common code is possibly the strongest evidence of all for the common ancestry of all life on Earth

Transcription Transcription is the DNA-directed synthesis of RNA
RNA synthesis Is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

Transcription I

Transcription II Exons = coding Introns = non-coding sequences
seqeunces

Transcription RNA forms base pairs with DNA
C-G A-U Primary transcript - length of RNA that results from the process of transcription

RNA RNA is single stranded, not double stranded like DNA
RNA is short, only 1 gene long, where DNA is very long and contains many genes RNA uses the sugar ribose instead of deoxyribose in DNA RNA uses the base uracil (U) instead of thymine (T) in DNA

Major Players in Transcription
mRNA - type of RNA that encodes information for the synthesis of proteins and carries it to a ribosome from the nucleus

Major Players in Transcription
RNA polymerase - complex of enzymes with 2 functions: Unwind DNA sequence Produce primary transcript by stringing together the chain of RNA nucleotides

Transcription RNA Polymerase binds to a special sequence of DNA called a Promoter RNA Polymerase opens up the DNA Helix RNA Polymerase uses one side of the DNA as a Pattern from which to form an RNA strand Complementary Base Pairing Allows the Correct RNA Nucleotide to be placed in position Then RNA Polymerase attaches two nucleotides together to start forming the RNA

Transcription As Transcription proceeds along the gene, the DNA molecule will rewind behind the RNA Polymerase The new RNA molecule is then displaced

Transcription As Transcription Proceeds along the gene, the DNA molecule will rewind behind the RNA Polymerase The new RNA molecule is then displaced Finally the end of the gene is reached and the RNA Polymerase comes off of the DNA The RNA “Transcript” is completely displaced from the DNA The Initiation Stage of Transciption is heavily regulated by cells

Synthesis of RNA Transcript - Initiation
TRANSCRIPTION RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide T A TATA box Start point Template DNA strand 5 3 Transcription factors Promoter RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Eukaryotic promoters 1 Several transcription 2 Additional transcription 3 Promoters signal the initiation of RNA synthesis Transcription factors help eukaryotic RNA polymerase recognize promoter sequences

Synthesis of RNA Transcript - Elongation
RNA polymerase synthesizes a single strand of RNA against the DNA template strand (anti-sense strand), adding nucleotides to the 3’ end of the RNA chain As RNA polymerase moves along the DNA it continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides Elongation RNA polymerase Non-template strand of DNA RNA nucleotides 3 end C A E G U T 3 5 Newly made Direction of transcription (“downstream”) Template

Synthesis of an RNA Transcript - Termination
Specific sequences in the DNA signal termination of transcription When one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again

Transcription Overview

Primary Transcript Modifications for mRNA
5’ CAP Removal of Intervening/Intruding Sequences (Introns) Polyadenylated Tail

mRNA Processing Primary transcript is not mature mRNA
DNA sequence has coding regions (exons) and non-coding regions (introns) Introns must be removed before primary transcript is mRNA and can leave nucleus

Post Termination mRNA Processing
Most eukaryotic mRNAs aren’t ready to be translated into protein directly after being transcribed from DNA. mRNA requires processing. Transcription of RNA processing occur in the nucleus. After this, the messenger RNA moves to the cytoplasm for translation. The cell adds a protective cap to one end, and a tail of A’s to the other end. These both function to protect the RNA from enzymes that would degrade Most of the genome consists of non-coding regions called introns Non-coding regions may have specific chromosomal functions or have regulatory purposes Introns also allow for alternative RNA splicing Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things: Add protective bases to the ends Cut out the introns

Alteration of mRNA Ends
Each end of a pre-mRNA molecule is modified in a particular way The 5 end receives a modified nucleotide cap The 3 end gets a poly-A tail A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end Protein-coding segment Polyadenylation signal Poly-A tail 3 UTR Stop codon Start codon 5 Cap 5 UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P 5 3

RNA Processing - Splicing
The original transcript from the DNA is called pre-mRNA It contains transcripts of both introns and exons The introns are removed by a process called splicing to produce messenger RNA (mRNA)

RNA Processing Proteins often have a modular architecture consisting of discrete structural and functional regions called domains In many cases different exons code for the different domains in a protein Gene DNA Exon 1 Intron Exon 2 Exon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide

Transcription is done … what now?
Now we have mature mRNA transcribed from the cell’s DNA. It is leaving the nucleus through a nuclear pore. Once in the cytoplasm, it finds a ribosome so that translation can begin. We know how mRNA is made, but how do we “read” the code?

Translation Second stage of protein production
mRNA imoves to and binds with a ribosome

Ribosomes 2 subunits, separate in cytoplasm until they join to begin translation Large Small Contain 3 binding sites E P A

Translation Second stage of protein production mRNA is on a ribosome
tRNAs bring amino acids to the ribosome

Translation at the Ribosome

Translation Translation is the RNA directed synthesis of a polypeptide
TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly A G C U Codons 5 3 Translation is the RNA directed synthesis of a polypeptide Translation involves mRNA: Genetic coding by triplet codons Ribosomes - Ribosomal RNAs & Ribosomal proteins Transfer RNAs

Translation Requires mRNA, tRNA, and rRNA
The Genetic Code are the rules by which a set of 3 nucleotides on the mRNA (a codon) determine a particular Amino Acid Note there are 20 amino acids and some amino acids are designated by more than one codon

tRNA Function Amino acids must be in the correct order for the protein to function correctly tRNA lines up amino acids using mRNA code

tRNA Transfer RNA Bound to one amino acid on one end
Anticodon on the middle loop complements the mRNA codon

Translation Transfer RNA molecules carry individual amino acids to the site of protein synthesis T RNAs have a sequence of 3 nucleotides that complementary base pairs with the codon = the anticodon

Translation The Ribosome is composed of rRNA and Ribosomal Proteins
It is the site where mRNA & tRNAs come together to build a polypeptide chain The ribosome has the enzymatic activity that catalyzes peptide bond formation between one amino acid and the next as a protein is built

Which Codons Code for which Amino Acids?
Genetic code - inventory of linkages between nucleotide triplets and the amino acids they code for A gene is a segment of RNA that brings about transcription of a segment of RNA

The Genetic Code The code is universal because it specifies the same 20 amino acids in all organisms with only few exceptions The code is redundant or degenerate because there are multiple codons which code for the same AA The code is unambiguous because any one codon codes for only one amino acid The code is a triplet code because 3 bases represent a single AA in a codon

The Genetic Code Genetic information is encoded as a sequence of nonoverlapping base triplets, or codons DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION mRNA Protein TRANSLATION Amino acid A C G T U Trp Phe Gly Ser Codon 3 5

How many bases code for each amino acid?
1 base = 1 amino acid 41 = 4 2 bases = 1 amino acid 42 = 16 3 bases = 1 amino acid 43 = 64

Reading the DNA code Every 3 DNA bases pairs with 3 mRNA bases
Every group of 3 mRNA bases encodes a single amino acid Codon - coding triplet of mRNA bases

The Genetic Code Codons: 3 base code for the production of a specific amino acid, sequence of three of the four different nucleotides Since there are 4 bases and 3 positions in each codon, there are 4 x 4 x 4 = 64 possible triplet codons 64 codons but only 20 amino acids, therefore most have more than 1 codon / the code is redundant 3 of the 64 codons are used as STOP signals; they are found at the end of every gene and mark the end of the protein One codon is used as a START signal: it is at the start of every protein Universal: in all living organisms

The Genetic Code A codon in messenger RNA is either translated into an amino acid or serves as a translational start/stop signal Second mRNA base U C A G UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Met or start Phe Leu lle Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UGU UGC Tyr Cys CAU CAC CAA CAG CGU CGC CGA CGG AAU AAC AAA AAG AGU AGC AGA AGG GAU GAC GAA GAG GGU GGC GGA GGG UGG UAA UAG Stop UGA Trp His Gln Asn Lys Asp Arg Gly First mRNA base (5 end) Third mRNA base (3 end) Glu

Transfer RNA Consists of a single RNA strand that is only about 80 nucleotides long Each carries a specific amino acid on one end and has an anticodon on the other end A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids tRNA brings the amino acids to the ribosomes Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) (a) 3 C A G U * 5 Amino acid attachment site Hydrogen bonds Anticodon The “anticodon” is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule

Transfer RNA 3 dimensional tRNA molecule is roughly “L” shaped 5 3 A
(b) Three-dimensional structure Symbol used in the book Amino acid attachment site Hydrogen bonds Anticodon A G 5 3 (c)

Ribosomes Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis The 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Exit tunnel Growing polypeptide tRNA molecules E P A Large subunit Small Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. (a) 5 3

Building a Polypeptide
Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA mRNA Codons 3 5 Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. (c)

Building a Polypeptide
We can divide translation into three stages Initiation / Elongation / Termination The AUG start codon is recognized by methionyl-tRNA or Met Once the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chain RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain Translation ends when a stop codon (UAA, UAG, UGA) is reached

Translation Initiation

Initiation of Translation
The initiation stage of translation brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome Large ribosomal subunit The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid. 2 Initiator tRNA mRNA mRNA binding site Small Translation initiation complex P site GDP GTP Start codon A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). 1 Met U A C G E 3 5

Translation Elongation

Elongation of the Polypeptide Chain
In the elongation stage, amino acids are added one by one to the preceding amino acid Amino end of polypeptide mRNA Ribosome ready for next aminoacyl tRNA E P A GDP GTP 2 site 5 3 TRANSCRIPTION TRANSLATION DNA Ribosome Polypeptide Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 1 Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3

The Formation of Peptide Bonds

Translation Termination

Termination of Translation
The final stage is termination when the ribosome reaches a stop codon in the mRNA Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 5 3 When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. 1 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. 2 3 The two ribosomal subunits and the other components of the assembly dissociate.

Translation The final step in translation is termination. When the ribosome reaches a STOP codon, there is no corresponding transfer RNA Instead, a small protein called a “release factor” attaches to the stop codon The release factor causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide The messenger RNA can be translated many times, to produce many protein copies

Translation in Eukaryotes
Occurs on free ribosomes in the cytosol Or can occur while ribosomes are attached to the rough endoplasmic reticulum (rough ER)

A Summary of Transcription and Translation in a Eukaryotic Cell
RNA is transcribed from a DNA template. DNA RNA polymerase transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. Exon Poly-A RNA transcript (pre-mRNA) Intron NUCLEUS Cap FORMATION OF INITIATION COMPLEX After leaving the nucleus, mRNA attaches to the ribosome. CYTOPLASM mRNA Growing polypeptide Ribosomal subunits Aminoacyl-tRNA synthetase Amino acid tRNA AMINO ACID ACTIVATION Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Activated amino acid TRANSLATION A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) Anticodon A C U G E Ribosome 1 5 3 Codon 2 3 4 5 Figure 17.26

Translation

Post-Translation The new polypeptide is now floating loose in the cytoplasm if translated by a free ribosme. It might also be inserted into a membrane, if translated by a ribosome bound to the endoplasmic reticulum. Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins. Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.

Transcription vs. Translation Review
Process by which genetic information encoded in DNA is copied onto messenger RNA Occurs in the nucleus DNA mRNA Translation Process by which information encoded in mRNA is used to assemble a protein at a ribosome Occurs on a Ribosome mRNA protein

Chromosomes

Chromosomes A eukaryotic chromosome is a molecule of DNA together with associated proteins Chromosome Structure made of DNA and associated proteins Carries part or all of a cell’s genetic information

Chromosome Structure Sister chromatid Centromere
One of two attached members of a duplicated eukaryotic chromosome Centromere Constricted region in a eukaryotic chromosome where sister chromatids are attached

Chromosome Structure Proteins organize DNA structurally Histones
Allow chromosomes to pack tightly Histones Type of protein that structurally organizes eukaryotic chromosomes Nucleosome A length of DNA wound around a spool of histone proteins

Chromatin in Eukaryotes
Grainy threadlike material seen in the nucleus DNA molecules organize into large, compact visible chromosomes before each cell division Chromosomes contain DNA & coiling proteins DNA is first wrapped around histone proteins - "beads on a string” Higher levels of DNA packaging (supercoiling)

Chromatin Organization
DNA is packaged by various levels of supercoiling

Chromatin Organization

Chromosome Structure

Mutations

Mutations Uncorrected errors in DNA replication may become mutations
A permanent change in DNA sequence

Variation: Central Questions
What is the relationship between the genetic variation of the genotype and variation of the phenotype? genotype + gene regulation + gene interactions within the genome + developmental processes + environmental effects and constraints + random-variation → phenotype

Variation: Central Questions
What are the mechanisms by which mutations and modifications of gene regulation serve as sources of variation?

Mutations Have Many Causes
Spontaneous Mutations DNA copy errors Induced Mutations Mutagens Radiation Viruses Transposons Somatic Mutations Germline Mutations Mutations and Health A Service of the U.S. National Library of Medicine POLYPOLOIDY (ENTIRE SETS OF CHROMOSOMES UK Science Museum Short Animation

Point Mutations Point mutations involve alterations in the structure or location of a single gene. Generally, only one or a few base pairs are involved. Point mutations can significantly affect protein structure and function Point mutations may be caused by physical damage to the DNA from radiation or chemicals, or may occur spontaneously Point mutations are often caused by mutagens

Mutations in the Genome
Point mutations can occur through: substitutions (change in bases) including tautomer errors insertions (introduction of bases) deletions (loss of bases) within the DNA or change in gene position: transpositions

Transitions and Transversions
Point Mutations Transitions outnumber Transversions 2:1

Tautomers of Nitrogenous Bases

Point Mutation The change of a single nucleotide in the DNA’s template strand leads to the production of an abnormal protein In the DNA, the mutant template strand has an A where the wild-type template has a T. The mutant mRNA has a U instead of an A in one codon. The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Mutant hemoglobin DNA Wild-type hemoglobin DNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val C T A G U 3 5

Types of Point Mutations
Point mutations within a gene can be divided into two general categories Base-pair substitutions Base-pair insertions or deletions

Mutation From a Functional Perspective
Sense Mutation: There is a change in the DNA base sequence but no change in amino acids in the polypeptide structure Missense Mutation: There is a change in the DNA base sequence, and a change in amino acids in the polypeptide structure, but the protein is still functional to some degree Nonsense Mutation: There is a change in the DNA base sequence and a change in amino acids in the polypeptide structure, and the protein is non-functional, a fragment, or is not produced at all

Point Mutations Silent or synonymous
Silent mutations are much more likely when the point mutation is in the third position of the codon triplet

Point Mutations Replacement or nonsynonymous Stop codon

Loss-of-Function Mutations
Insertion Deletion Stop Codon  Frameshift mutations 

Mutations A mismatching of base pairs, can occur at a rate of 1 per 10,000 bases DNA polymerase proofreads and repairs accidental mismatched pairs Chances of a mutation occurring at any one gene is over 1 in 100,000 Because the human genome is so large, even at this rate, mutations add up Each of us probably inherited 3-4 mutations!

Mutation Causes and Rate
The natural replication of DNA produces occasional errors. DNA polymerase has an editing mechanism that decreases the rate, but it still exists.\ Typically genes incur base substitutions about once in every 10,000 to 1,000,000 cells. Since we have about 6 billion bases of DNA in each cell, virtually every cell in your body contains several mutations. However, most mutations are neutral: have no effect. Only mutations in cells that become sperm or eggs—are passed on to future generations. Mutations in other body cells only cause trouble when they cause cancer or related diseases.

Mutations in the Genome
Major transposition of DNA segments can produce chromosomal inversions Segments of DNA can be rearranged to new locations and even to other chromosomes producing chromosomal translocations

Mutagens Mutagens are chemical or physical agents that interact with DNA to cause mutations. Physical agents include high-energy radiation like X-rays and ultraviolet light Chemical mutagens fall into several categories. Chemicals that are base analogues that may be substituted into DNA, but they pair incorrectly during DNA replication. Interference with DNA replication by inserting into DNA and distorting the double helix. Chemical changes in bases that change their pairing properties. Tests are often used as a preliminary screen of chemicals to identify those that may cause cancer Most carcinogens are mutagenic and most mutagens are carcinogenic.

Viral Mutagens Scientists have recognized a number of tumor viruses that cause cancer in various animals, including humans About 15% of human cancers are caused by viral infections that disrupt normal control of cell division All tumor viruses transform cells into cancer cells through the integration of viral nucleic acid into host cell DNA

End of Review

DNA Structure and Function

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