Molekler Genetik I (MBG 222) Yrd. Do. Dr.

Ayten Kandilci
letiim bilgisi: akandilci@gyte.edu.tr E-mail : Alttan alnan dersler belirtilsin. letiim e-mail aracl ile salanacak
Kaynaklar:

* 1.Watson, J.D., et al. Molecular Biology of the Gene. 6/E The Benjamin/
Cummings Pub. Co., Menlo Park, California, 2008.

* 2.Alberts, B., et al. Molecular Biology of the Cell. 5/E. ed. Garland Pub., New York,
2008.

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Chapter 2 Nucleic Acids Convey Genetic Information

Outline
DNA Can Carry Genetic Specificity

The Double Helix The genetic information within the DNA is conveyed by
the sequence of its four nucleotide building blocks The Central Dogma Establishing the direction of protein synthesis

The era of genomics

TRANSFORMATION FROM NONVIRULENCE TO VIRULENCE IS HEREDITARY

Frederick Griffith (British microbiologist) 1928: Non virulent strains of pneumoniae causing bacteria (Streptococcus pneumoniae) become virulent when mixed with their heat-killed pathogenic counterparts. This transformation was hereditary and passed to descendants of newly pathogenic strains. Without knowing whether or not subsequent generations were also virulent, it was also possible to conclude that the cells became virulent because they received a factor directly responsible for virulence, rather than the genetic information coding for the factor. In that case, however, the virulence factor would have been diluted upon division, and would not have been present in subsequent generations

Frederick Griffith (1879-1941)

TRANSFORMATION FROM NONVIRULENCE TO VIRULENCE IS HEREDITARY

Griffiths experiment raised the possibility that when pathogenic cells are killed by heat:
A) Their genetic components remain undamaged, B) These components pass through the wall of recipient cells and recombine with recipients genetic apparatus. (Fred Griffith. The Significance of Pneumococcal Types. Journal of Hygiene (1928), 27 : pp 113159 )

Frederick Griffith (1879-1941)

TRANSFORMATION FROM NONVIRULENCE TO VIRULENCE IS HEREDITARY

Figure 2-1: Transformation of a genetic characteristic of a bacterial cell by addition of heat-killed cells of a genetically different strain.

Active Genetic Principle in S. pneumonia is DNA

Oswald T. Avery, Colin MacLeod and Maclyn McCarty :
1944: Transforming activity of purified active fractions was destroyed by deoxyribonuclease (DNase; An enzyme that degrades DNA but has no effect on proteins or RNA). Addition of ribonuclease (RNase) or several proteolytic enzymes had no effect of transforming activity. Active genetic component that causes transformation was DNA. (Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty. J Exp Med. 1944 February 1; 79(2): 137158. )
Oswald Theodore Avery (1877-1955) (Canadian-American microbiologist) Colin MacLeod (1909-1972) (Canadian-American geneticist)

Isolation of a chemically pure transforming agent

(figure 2-2)

Viral Genes Are Also Nucleic Acids

Alfred D.Hershey and Martha Chase (Cold Spring Harbor Laboratory, Long Island, USA);
1952: They labeled the coat and the DNA of bacteriophage T2 with different radioactive isotopes to see which part of labeled atom of parental phage entered the host cell and later appeared in the progeny phage.

Much of the parental nucleic acid but none of the parental protein was detected in the progeny phage.
Result: Only the DNA component of the bacteriophage T2 carries the genetic information and the protein coat serves as a protective shell.
(Bacteriophage or phage: Bacterial virus)

The Double Helix

1938: First X-ray diffraction pattern of DNA (1938, William Astbury). 1952-1953: High quality X-ray diffraction photographs of DNA (Rosalind Franklin, Maurice Wilkins )
Suggested that DNA structure is helical and it is composed of more than one polynucleotide chain.
The key X-ray photograph involved in elucidation of DNA structure (Taken by Rosalind Franklin) (Fig.2-4)

Chargaffs Rule
1949: Erwin Chargaff (Biochemist); Examined the relative proportions of adenine, cytosine, guanine, and thymine in DNA . He Discovered that:
a. the different nucleotides were not all present at the same concentrations in DNA; b. their relative levels differed in different organisms; c. and that adenosine and thymine, and cytosine and guanine, are present at similar levels (A=T and G=C).

Phosphodiester bonds link together the nucleotides of DNA

1952: Alexander Todds group showed that 3-5 phosphodiester bonds link together the nucleotides of DNA.

The Double Helix

1953: Francis H. Crick and James D. Watson discovered the double-helical structure of DNA (published in Nature, April 25, 1953).

Francis H. Crick (1916-2004)

James D. Watson (1928- )

Crick, Watson and Maurice Wilkins awarded the 1962 Nobel Prize for Physiology or Medicine, "for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material."

Finding The Polymerase That Make DNA

1956: Arthur Kornberg had demonstrated DNA synthesis in cellfree extracts of bacteria. He showed that a specific polymerizing enzyme was needed to catalyze the linking of precursors of DNA. DNA precursors (nucleotide building blocks of DNA): -dATP (deoxyadenosine-triphosphate) -dTTP (deoxythymidine-triphosphate) -dCTP (deoxycytidine-triphosphate) -dGTP (deoxyguanosine-triphosphate)

Figure 2-7: The nucleotides of DNA

Enzymatic synthesis of a DNA chain catalyzed by DNA Pol I

The polimerizing enzyme DNA polymerase I (DNA Pol I) links the nucleotides by 3-5phosphodiester bonds. DNA Pol I depends on a DNA template to determine the sequence of the DNA it is synthesizing.

Figure 2-8

Proposed models of DNA replication

A) Dispersive (non-conservative model): DNA strand is broken in small pieces and used to prime for synthesis of new DNA. Than these pieces are joined together. B) Semiconservative model: Single strand of DNA is conserved during replication and each parental strand is distributed into each of the two daughter strands.

C) Conservative model : Both of the parental strains remain together and the two new strands of DNA form an entirely new DNA molecule.

DNA strands separate from each other during replication

1958: Matthew Meselson and Franklin W. Stahl:
They labelled the parental and daughter DNA with heavy 15N and light 14N isotopes, respectively. Separated the heavy (15N- 15N), light (14N- 14N) and heavy+light (15N-14N) hybrid DNA by centrifugation in density gradients of heavy salt cesium cloride. This experiment showed that double helix permanently separate from each other during replication and DNA replication is semiconservative.

DNA replication is a semiconservative process

A) Dispersive (non-conservative model): DNA strand is broken in small pieces and used to prime for synthesis of new DNA. Than these pieces are joined together. B) Semiconservative model: Single strand of DNA is conserved during replication and each parental strand is distributed into each of the two daughter strands.

C) Conservative model : Both of the parental strains remain together and the two new strands of DNA form an entirely new DNA molecule.

Evidence That Genes Control Amino Acid Sequences in Proteins

Wild-type (WT) hemaglobin (Hb) molecules contains alpha and beta globin chains, which are encoded by different genes. Sickle-cell anemia: Individuals have beta-globinS allele. 1957: Vernon M. Ingram showed that S-Hb differs from normal Hb only by one amino acid (aa). Because this change in aa sequence was observed only in patients with the S-allele of the gene, it was hypothesized S-allele of the gene encodes the change in the beta globin gene.

DNA Cannot Be the Template That Directly Orders Amino Acids During Protein Synthesis In eukaryotic cells; Protein synthesis occurs at sites where DNA is absent Protein synthesis in all eukaryotic cells occurs in the cytoplasm, which separated by nuclear membrane from the chromosomal DNA. Therefore, at least for the eukaryotic cells, a second information-containing molecule had to exist that obtains its genetic specifisity from DNA. This molecule would then move to cytoplasm to function as the template for protein synthesis.

The Central Dogma

Duplication Translation Transcription DNA RNA Protein

1953: The working hypothesis was adopted that chromosomal DNA functions as the template for RNA molecules, which subsequently move to cytoplasm, where they determine the arrangement of amino acids within protein.
1956: Francis Crick referred to this pathway for the flow of genetic information as the central dogma.

Discovery of transfer RNA (tRNA)

The discovery of how proteins are synthesized required the development of cell-free extracts capable of making proteins from amino acids.
These were first effectively developed beginning in 1953 by Paul C. Zamecnik and his collaborators. They discovered that prior to their incorporation into proteins, amino acids are first attached to what we now call transfer RNA (tRNA). tRNA accounts for about 10% of all cellular RNA.
Figure 2-14: Yeast alanine tRNA structure

Discovery of Messenger RNA (mRNA)

(Only a few percent of cellular RNA is mRNA)
Bacteria infected with phage T4 provided the ideal system to find the true template for protein synthesis.

E.Coli cells infected with phage T4 stop synthesizing E. Coli RNA; the only RNA synthesized is transcribed off the T4 DNA.
This T4-RNA does not contribute to ribosomal structure but it attaches and move across the ribosomal surface to bring its bases into positions where they can bind to tRNA-amino acid precursors for protein synthesis. T4 RNA orders the amino acids; thus it is the template for protein synthesis. Since it carries the information from DNA to proteins, it is called messenger RNA (mRNA).
Figure 2-15: Transcription and translation

Enzymatic Synthesis of RNA upon DNA template is Catalyzed by RNA Polymerase

1960: Jerard Hurwitz and Samuel B. Weiss discovered the RNA polymerase.
It synthesize RNA upon DNA template using RNA precursors: ATP, UTP, GTP and CTP. In every enzymatic synthesis, the RNA AU/GC ratio is similar to the AT/GC ratio of the template DNA (evidence that DNA lines up the correct ribonucleotide precursors).

Synthesis of RNA always begins at 5 end and concludes with the 3-end nucleotide.

RNA IS SYNTHESIZED IN THE NUCLEUS AND MOVES TO THE CYTOPLASM

Evidence for the postulated movement of RNA from the DNAcontaining nucleus to the ribosome-containing cytoplasm came from the following experiment:
Cells were exposed to radiactive cytidine for 12 minutes with (a pulse chase experiment), Then allowed to grow for 88 minutes in the presence of excess amount of unlabeled ribonucleotides (with this experiment, mRNA synthesized during a short time period was labeled).

Establishing the Genetic Code

1960s (Charles Yanofsky and Sydney Brenner): Successive group of nucleotides along a DNA chain code for successive amino acids along a given polypeptide.
1961 (Brenner and Crick): Groups of three nucleotides are used to specify individual amino acids.

Establishing the Genetic Code

Which specific groups of three bases (codons) determine which specific amino acids?

1961 (Marshall Nirenberg and Heinrich Matthaei): Addition of polynucleotide poly U (UUUUUU) to a cell free system capable of making proteins leads to the synthesis of polypeptide chains containing only the phenyl alanine The nucleotide group UUU must specify phenyl alanine.
Completion of the code in 1966 showed that 61 out of 64 permuted groups (43) corresponds to amino acids (aa), and most aa are encoded by more than one nucleotide triplet.

START AND STOP SIGNALS OF TRANSLATION ARE ALSO ENCODED WITHIN DNA

Translation both starts and stops at internal positions in mRNA

These start and stop signals that initiate and terminate translation are present within DNA (and its mRNA products):

UAA, UAG, UGA (non sense codons or STOP codons)

AUG (methionin, START codon) In prokaryotes, AUG codons that start new polypeptide chains are preceded by specific purin-rich blocks of nucleotides that serve to attach mRNA to ribosomes. In eukaryotes, the position of AUG relative to the beginning of the mRNA is critical determinant, and first AUG is always selected as the start site of translation.