Nucleic Acid Conveys Genetic Information

transformation

describe the insertion of new genetic material into bacterial or nonbacterial cells, including animal and plant cells

tRNA

1. amino acids attach to these prior to incorporation into protein 2. accounts for about 10% of all cellular RNA 3. contains a sequence of adjacent bases (anticodon) that bind specifically during protein synthesis to successive groups of bases (codons) along RNA template

experimental evidence for semiconservative model

1. bacteria grown in medium with heavy isotope (N15) 2. then transferred to light medium (N14) 3. heavy DNA was separated from light DNA in density gradients of heavy salts after certain amount of time using centrifugal force

nucleotide building blocks for DNA

1. dATP 2. dCTP 3. dGTP 4. dTTP

reading of single mRNA by multiple ribosomes

1. each ribosome attaches at a start signal along 5' end of mRNA chain and synthesize a peptide as it proceeds along the chain 2. several ribosomes may be attached to one mRNA molecule at a time

pulse chase experiments to monitor mRNA synthesis

1. exposed cells to radioactively labeled precursors and added a large number of unlabeled ribonucleotides 2. studies showed that mRNA is synthesized in nucleus, leaves within an hour and seen in cytoplasm

rRNA

1. originally thought to be template for ordering amino acids 2. neither small nor large class of this had feel of template RNA

pulse chase experiment to see how polynucleotide chains direct synthesis of polypeptides

1. provided b-globin RNA in cell-free system 2. cell-free system treated with radioactive amino acid, after which protein synthesis was immediately stopped 3. isolated only complete proteins via gel electrophoresis 4. partially cleaved proteins at particular site 5. proteins last to be synthesized would have highest density of radiolabeled precursors

rRNA functions

1. serve as factories for protein synthesis 2. bring tRNA into positions where they can read off the information provided by mRNA templates

evidence that genes control amino acid sequences in proteins

1. sickle-cell anemia characterized by red blood cells have sickle shape 2. if both B globin gene alleles are S form, it is most severe 3. wild-type hemoglobin molecules are constructed from alpha and B chains 4. sickle hemoglobins vary from normal hemoglobin by change of one amino acid at B-globin chain 5. glutamic acid replaced by valine at position 6

differences between RNA and DNA

1. sugar is ribose (has additional OH group) 2. RNA has uracil (not thymine) 3. uracil can form bond with A and G-not as specific as DNA T

conclusion of experiment regarding direction of protein synthesis

1. the direction is from the amino terminus to the carboxyl terminus 2. the new amino acid is added to the carboxyl terminus of the growing polypeptide chain

Avery's bombshell

DNA can carry genetic specificity

similarities between RNA and DNA

1. long, unbranched molecule 2. has four types of nucleotides linked together by 3'-5' phosphodiester bonds 3. can be synthesized on DNA template

human genome

1. more than 3 billion base pairs 2. encodes more than 20,000 genes

historical background regarding DNA carrying genetic information

1. nuclei isolated from pus in 1869 2. found nuclein, which is mostly chromatin 3. DNA and RNA were separated from proteins in late 1800s

why was mRNA overlooked as template

1. only a few percent of total cellular RNA is mRNA 2. has expected large variations in length and nucleotide composition required to encode different proteins found in cells

conservative model

Both parental strands stay together after DNA replication

slight issue with central dogma

RNA chains sometimes do act as templates for DNA chains of complementary sequences

Avery's contribution

identified transforming principle as DNA -- properties of transforming principle destroyed by DNAase (enzyme!), but not by protein destroying enzymes

stop codons

1. UAA 2. UAG 3. UGA

Avery's experiment steps

1. break cells 2. isolate DNA 3. add DNA to R cells 4. recombination and cell division 5. overall he isolated DNA, then destroyed DNA and proteins to compare what happened

X-ray diffraction pattern of DNA

1. confirmed that DNA is helical 2. purine and pyrimidine bases are regularly stacked next to each other, perpendicular to the helical axis 3. confirmed DNA made of more than 1 polynucleotide chain

Kornburn

1. demonstrated DNA synthesis in cell-free extracts of bacteria 2. revealed nucleotide building blocks for DNA are energy-rich precursors 3. showed DNA Polymerase I was capable of catalyzing synthesis of new DNA strands

possible mechanisms for DNA replication

1. dispersive model 2. semiconservative model 3. conservative model

semiconservative model

Type of DNA replication in which the replicated double helix consists of one old strand, derived from the old molecule, and one newly made strand

chromatin

a complex of DNA and chromosomal proteins

Griffith's Transformation Experiment results

when pathogenic cells were killed by heat, their genetic components were undamaged

How is RNA made

1. RNA polymerase enzymes function only in presence of DNA 2. DNA serves as template upon which single-stranded RNA chains are made 3. only one of two strands of DNA is used as template to make RNA during transcription

Chargaff's rule

1. There is always equal amounts of adenine with thymine, and equal amounts of guanine and cytosine 2. A=T and G=C

Meselson and Stahl prediction

1. all DNA molecules will have one heavy and one light strand after 1 generation 2. half DNA molecules will have light strands and half will have hybrid strands after 2 generations

issues with rRNA acting as template

1. all E. coli ribosomes are composed of two unequally sized subunits, each containing RNA that can stick together or fall apart in a reversible manner 2. rRNAs in subunits are of similar chains 3. base composition of both small and large rRNA chains are approximately the same

bacterial virus

1. also called bacteriophage 2. contains DNA core and protective shell made of proteins

direction of RNA synthesis

1. always proceeds in a fixed direction 2. beginning at 5' end and concluding at 3' end

translational start codons

1. amino acid methionine initiates all polypeptide chains 2. AUG codes for initiating methionine and also codes for methionine residues 3. prokaryotes: AUG codons that start new polypeptide preceded by specific purine rich nucleotides 4. eukaryotes: position of AUG relative to beginning of the mRNA is determinant-first AUG being selected as start site of translation

implications of discovery of a double helix

1. gene no longer a mystery 2. intertwined strands of complementary structures suggested that one strand serves as template upon which the other strand is made (semiconservative replication)

Confirmation for DNA as genetic material

1. geneticists doubted use of DNA as genetic material as it appeared to be monotonous repeats of 4 bases 2. Watson and Crick published double-helical model of DNA structure 3. Chargaff demonstrated that 4 bases not present in equal proportions 4. Hershey and Chase demonstrated that bacteriophage infection comes from DNA

establishing the genetic code

1. groups of several nucleotides must specify a given amino acid because there are 20 amino acids and only 4 bases 2. genetic study found groups of three nucleotides are used 3. biochemical analysis using synthetic polynucleotides in a cell-free system capable of making proteins found that UUU contains only phenylaline

insights into DNA sequence provided by comparative genomics

1. identification of DNA sequence that direct gene expression 2. DNA replication 3. chromosome segregation 4. recombination 5. identify mutations that lead to disease

discovery of mRNA

1. infected cells with T4 phage 2. following infection, cells stopped synthesizing its own RNA and only synthesized from T4 DNA 3. T4 RNA has base composition similar to T4 DNA and doesn't bind to ribosomal proteins that normally associated with rRNA to form ribosomes 4. T4 RNA orders the amino acid and is template

ideas that led to adaptor hypothesis

1. specific chemical groups on 4 bases of RNA should mostly interact with water-soluble groups, while many specific amino acids prefer to interact with water insoluble groups 2. it is unlikely that RNA template can discriminate between chemically similar amino acid groups

Discovery of tRNA

1. this discovery required the development of cell-free extracts capable of making amino acid precursors as directed by added RNA molecules 2. used radioactively tagged amino acids to trace newly made proteins and combined that with preparative centrifuge to fractions everything found to be engaged in protein synthesis

Griffith's Transformation Experiment

A deadly bacteria transformed a harmless bacteria, causing it to be deadly, the experiment proved that information could change cells, and the information could be passed to the next generation

polyribosome

An aggregation of several ribosomes attached to one messenger RNA molecule.

Adaptor hypothesis

amino acids are first attached to specific adaptor molecules, which in turn possess unique surfaces that can bind specifically to bases on the RNA templates

Hershey and Chase results

much of parental nucleic acid and none of the parental protein was detected in the progeny phage

translation start/stop locations

occurs at internal positions

what links nucleotides in DNA

1. 3'-5' phosphodiester bonds 2. nucleotides connected by covalent bonds

how does DNA genetic information work to order amino acids during protein synthesis

1. DNA is built up of 4 bases 2. potential sequences for a fragment with N nucleotide is 4^N

Double helix model

1. DNA is double helix with sugar-phosphate backbone on outside and bases aligned on interior 2. 10 base pairs per turn 3. two stranded are antiparallel

dispersive model

1. DNA strands broken as frequently as every 10 base pairs and used to prime the synthesis of similarly short regions of DNA 2.Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA

experiments carried out on nucleic acids

1. Griffith bacteria transformation 2. Avery found DNA was genetic principle 3. Hershey and Chase: found nucleic acid carried genes 4. Wilkins and Franklin: X-ray diffraction 5. Watson and Crick: double helix 6. Meselson and Stahl: showed two strands separate during replication (semiconservative) 7. pulse labeleling to determine growing polypeptide chain direction

Meselson and Stahl results

DNA replication is semi conservative

Hershey and Chase experiment

They tagged bacteria phages with radioactive elements (sulfur for protein and phosphorus for DNA) to see which was the transforming factor of pneumonia

how to identify important parts of a protein

compare predicted amino acid sequences encoded by similar genes from different organisms

DNA can't be template that directly orders amino acids during protein synthesis

protein synthesis occurs at sites where DNA is absent-the cytoplasm

Avery's results

the capsule gene codes for a key enzyme involved in the synthesis of the carbohydrate-containing capsule that surrounds most pneumonia-causing bacteria