dna & rna structure and functiontriplets specify amino acids • code determined by nirenberg...

DNA & RNA STRUCTURE AND

FUNCTION

The search for the genetic material led to DNA

Introduction

• Although DNA was isolated in 1869 by Meischer.....

• Turning point question that led to the history of molecular genetics occurred in the early 1940’s.

• Scientists’ opposing views

The ability of DNA(as genetic material) to cause bacterial transformation

Work of Griffith and Avery

Work of Griffith and Avery

The role of phage DNA in viral reproduction

• Review viral replication

• http://www.courses.fas.harvard.edu/~biotext/animations/lyticcycle.html

The role of phage DNA(as genetic material) in viral reproduction

• Hershey & Chase experiment

Correlation between DNA content & chromosome duplication provides

further evidence

• Findings of Alfred Mirsky

• somatic cells contain equal amounts of DNA

• gametes contain 1/2 as much DNA as somatic cells

Charfaff’s study of nitrogenous bases

•Background -

•Two types of bases in DNA

•purines & pyrimidines

Three components of DNA nucleotide

• nitrogenous base

• sugar - deoxyribose

• phosphate

Chargaff analyzed purine and pyrimidine content

• DNA has equal number of adenine and thymine (A=T) and equal numbers of guanine and cytosine(G=C)

• proportions are the same in all cells of a given species but vary species to species.

Watson & Crick discover the double

helix by building models to conform to

data

Work of Linus Pauling

• proteins can be helical (maintained by H bonds)

• perhaps DNA is helical

Rosalind Franklin and Maurice Wilkins x-ray

crystallography

• Franklin was responsible for much of the research and discovery work that led to the understanding of the structure of deoxyribonucleic acid, DNA.

• X-ray studied indicated that DNA was helical and provided dimensions

• By November 1951 Wilkins had evidence that DNA in cells as well as purified DNA had a helical structure.

• Alex Stokes had solved the basic mathematics of helical diffraction theory and thought that Wilkins's x-ray diffraction data indicated a helical structure in DNA.

• Molecule must

• be heterogeneous & varied - to carry large amount of genetic information

• replicate readily & precisely - to pass information on

Watson and Crick Model

Twisted Ladder Analogy

• sides of ladder - sugar and phosphate

• rungs - base pairs

• base - bonded to sugar

• base to each other by H bonds

• base pairing - purine with pyrimidine

Evidence of Purine-Pyrimidine Pairing

• X-ray measurements showed distance between sides was 2nm

• 2 purines > 2 nm

• 2 pyrimidines < 2 nm

• 1 purine & 1 Pyrimidine = 2 nm

DNA Model & Variety

• Nucleotides can be assembled in any order & 1000’s of nucleotides long, so the possibility fir great variety exists.

Structural Basis for Complimentary Base Pairing

• Structurally only A with T (2 H bonds) and C with G (3 H bonds)

DNA Replication - base pairing allows DNA to serve as template

• the two strands separate

• each strand directs the synthesis of a new complementary strandpurine

DNA Replication

• rates

• eukaryotes - 50 nucleotides/sec

• prokaryotes - 500 nucleotides/sec

• short stretches with specific nucleotides

Initiation - Starts at origin of replication

Replication - initiation

• Proteins attach at origin of replication and separate strands opening up a replication bubble.

• At each end of the bubble is a replication fork where the strands unwind.

Replication - initiation• Proteins participate:

• helicase – untwist and separate strands.

• single-strand binding proteins – bind to the single strands to stabilize them.

Replication - initaition• Proteins participate:

• topisomerase – relieves the strain (caused by

unwinding) ahead of the replication fork by breaking, swiveling and rejoining strands.

Replication - initiation• the synthesis of RNA primer

• initial nucleotide chain which is a short stretch ( 5 - 10 nucleotides) of RNA

• synthesized by RNA primase

• DNA cannot initiate the synthesis of a polynucleotide (can only add to the end of an already existing chain that is base-paired to the template)

Replication - Elongation

• DNA polymerases catalyze the synthesis of new DNA by adding nucleoside (SBP) triphoshates.

Replication - Elongation• Antiparallel Elongation: must occur in 5--> 3

direction.

• Occurs because along the “leading strand” DNA polymerse nestles in the replication fork and continuously adds nucleotides to the new complementary strand.

• Sliding clamp facilitates the process (protein that encircles newly synthesized helix like a doughnut and moves DNA polymerase along template).

Replication - Elongation• Synthesis of “lagging strand” occurs in the

opposite direction, to accommodate the 5-->3 direction :

• The lagging strand is synthesized as a series of fragments, Okazaki fragments.

• DNA ligase binds the Okazki fragments

Summary of Replication

• http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html


• occurs bidirectionally - 2 replication forks move in opposite directions away from the origin.

• semi-conservative - 2 new helixes consist of 1 new & 1 old strand

• number of replication origins:

• prokaryotes - single

• eukaryotes - many; bubbles expand bidirectionally and merge (chromosome has duplicated)


• DNA polymerase proofreads and repairs:

• will only add nucleotides if the preceding are correct

• if incorrect - backtracks and removes incorrect nucleotides

• VALUE - insures accuracy of replication.


• Enzymes constantly monitor DNA to maintain integrity

• If there is an incorrect nucleotide:

• enzymes move in, snip it out and replace it with the correct nucleotide.

Summary of Replication• Energetics of replication: nucleotides are added as

triphosphates (dATP,dCTP,dGTP,dTTP)

• As nucleotides are added, the 2~P’s are removed and power DNA polymerase

DNA as a carrier of Information

• Information carried in sequence of bases

• 5000 (viruses) to 6 billion (humans)

• library of 1000 books

The Genetic Code & Its Translation

• Garrod’s hypothesis in 1909

• Diseases are the result of inborn errors of metabolism


• DNA controls metabolism by commanding cells to make specific enzymes & proteins.

• work of Beadle (1932) - used mutant eye color in Drosophila


• 1941 experiment of Beadle & Tatum with Neurospora led to “one gene - one-polypeptide hypothesis”.


• One gene - one enzyme led to one gene - one polypeptide because:

• many proteins are not just enzymes such as hormones, structural proteins, membrane proteins


• Conclusion

• The DNA molecule contains a coded message with instructions for biological structure & function

• These instructions are carried out by proteins, which also contain a highly specific biological language - amino acid sequence

Protein Synthesis

• Basic mechanism of reading and expressing genes is from DNA to RNA to Protein – referred to as the central dogma of biology.

Protein Synthesis - Background

• Nucleic acids and proteins are informational polymers:

• Sequencing of nucleotides & amino acids

• Both have specific sequences of monomers that convey information


• DNA/RNA

• monomers of the 4 nucloetides

• the 4 nucleotides are dependent on the nitrogenous base present

• genes are 100’s or 1000’s of nucleotides long with a specific sequence


• Proteins are monomers of the 20 different amino acids


• RNA - ribonucleic acid

• basic unit is nucleotide

• sugar is ribose

• uracil replaces the base thymine

• single strand


• 3 clues that RNA plays a role in linking DNA with amino acid sequencing

• cells making large amounts of protein are rich in RNA

• cells making large amount of protein have large numbers of ribosomes, which are rich in RNA


• 3 clues that RNA plays a role in linking DNA with amino acid sequencing

• Viral Studies:

• During viral duplication RNA synthesis occurs before production of the protein coat

• Some viruses only have RNA & protein, yet during reproduction, protein coats are produced.


• Messenger RNA is intermediate in information flow

• mRNA

• are copies (transcripts) of nucleotide sequences in DNA.

Transcription - synthesis of RNA under the direction of DNA

Transcription

• RNA polymerases pry the 2 strands of DNA apart and join RNA nucleotides as they base pair along the DNA template.

• RNA polymerases can only assemble in 5-->3 direction.

• Does not require primer.

Transcription - Steps

• Initiation

• RNA polymerase binds to a promoter (DNA sequence where RNA polymerase attaches and begins transcription).

• Transcription unit – stretch of DNA that is transcribed into RNA

• Initiation – DNA strands unwind and polymerase initiates RNA synthesis at the start point of the template strand.


• Elongation

• the polymerase moves downstream unwinding the DNA and elongating the transcript; the DNA reforms the helix.

• Notice the difference in eukaryotes – fig. 17.8: processing


• Termination – the RNA transcript is released and the RNA polymerase detaches from DNA.

• http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html

Transcription - Differences between prokaryotes & eukaryotes

• Prokaryotes have a termination signal

• Eukaryotes have a polyadenylation signal which is transcribed;

• eventually the RNA transcript is cut free, as pre-RNA, still needs processing.

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

• role of splicesomes

Triplets specify amino acids

• Mathematical Arguments for triplet code - 4 bases available to code for 20 amino acids


• code determined by Nirenberg & Matthaei 10 years after the model was determined by Watson and Crick.

• Worked with E.coli

• used “poly-u” synthesized by Ochoa

• had 20 test tubes with E.coli and “poly-u” (along with ribosomes, ATP, enzymes, amino acids and one labeled

• one test tube produced polypeptide chains of phenylalanine therefore UUU codes for phenylalanine

• All codes figured out in a similar manner


• Many amino acids have more than one codon

• The codons usually differ in the third nucleotide

• Some do not represent any amino acid and are stop signals.

• All organisms have the same code, the code evolved early.

Translation - synthesis of a polypeptide

• Vocabulary:

• Promoter - site of transcription

• specific nucleotide sequences of DNA that are the binding sites for RNA polymerase

• the start signals for RNA synthesis


•Vocabulary:

•Terminator - nucleotide sequences that are the stop signals for RNA synthesis


• Vocabulary: mRNA - summary

• synthesized by transcription

• 500 to 1000 nucleotides long

• single-stranded

• has codon

Translation - synthesis of a polypeptide - because information is “translated” from one language

(nucleic acids) to another(proteins.)

• Vocabulary

• rRNA

• transcribed in the nucleolus

• composes 2/3 of ribosome


• Vocabulary

• rRNA

• ribosome - has 2 sub-units

• smaller - binding site for mRNA

• larger - 2 binding sites for tRNA

• P site - peptide site

• A site - aminoacyl site

• E site - exit site


• Vocabulary

• tRNA

• transcribed in the nucleus

• 20 kinds (1 per amino acid)

• small, coverleaf shape


• tRNA special areas:

• attachment site for specific amino acid

• attachment site for mRNA (has anticodon) - at other side of the loop

• recognition site for enzyme aminoacyl-tRNA synthetase (at binding site - powers binding of amino acid and tRNA

Translation - 3 steps• Initiation

• formation of initiator complex

• small ribosomal sub-unit, mRNA, initiator tRNA

• energy from GTP

• initiator codon, anticodon, 1st amino acid

Translation - 3 steps• Elongation

• formation of first peptide bond

• 2nd tRNA in “A” site

• bond forms between 2 amino acids

• 1st into E site, 2nd into P site, 3rd into A site

• repeated over and over

Translation - 3 steps• Termination

• termination signals at the end of mRNA coding sequence

• no tRNA with the anticodon to match the codon

• therefore no tRNA’s can enter the “A” site

Translation - summary

http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter3/

Translation• Polyribosomes

• group of ribosomes reading the same mRNA

• formed if another ribosome can form an initiation complex with the freed “initiator” portion of a mRNA still being translated

• make possible the rapid copies of a polypeptide from the instructions carried by a single mRNA

Prokaryote Genomes

• Prokaryotes

• circular, folded DNA

• 4.7 million base pairs

• 1.4 mm long - 500 x longer than cell

• called nucleoid

• plasmids - smaller circular DNA molecules in some bacter

Eukaryotic Chromosomes

• Chromatin - material of chromosome

• includes DNA, protein, RNA

• DNA is roughly the length of the chromosome BUT it is coiled and folded.

Eukaryotic Chromosomes

• histones

• conserved histones

• nucleosomes

• condensed chromatin

• centromere

• chromatids

• telomeres

• satellite DNA

dna & rna structure and functiontriplets specify amino acids • code determined by nirenberg...

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