Chapter 16 & 17

These properties of the genetic code make it more fault-tolerant for point mutations.

A practical consequence of redundancy is that some errors in the genetic code cause only a silent mutation or an error that would not affect the protein because the hydrophilicity or hydrophobicity is maintained by equivalent substitution of amino acids; for example, • a codon of NUN (where N = any nucleotide) tends to code for hydrophobic amino acids. • NCN yields amino acid residues that are small in size and moderate in hydropathy; • NAN encodes average size hydrophilic residues. •These variable codes for amino acids are allowed because of modified bases in the first base of the anticodon of the tRNA, and the base-pair formed is called a wobble base pair.

Aminoacyl-tRNA synthetase

- ATP-dependent enzymes that covalently link amino acids to tRNAs - Specific for each amino acid and for the corresponding tRNA(s)

Where does the replication fork happen?

-Happening at many places along the molecule

Post-modern formulation:

.•One gene can actually code several different polypetides or RNA subunits, by the process of alternative splicing. .•Alternative splicing of different exon sequences in different combinations creates different types of related proteins. .•These may be tissue specific. .•Many different types of gene products are transcribed to RNA but not translated to protein (e.g., rRNA, tRNA, SARNA). .•Other types of RNA sequences also are important for development and regulation of gene expression (e.g., miRNAs, siRNA). .•Non-coding DNA sequences also function importantly in gene regulation (e.g., binding sites for transcription etc.). .•Ultimately, the # of genes in not so important. This # is remarkably conserved among species (e.g., Drosophila vs Human). How the genes are expressed is more essential.

DNA replication in prokaryotic and eukaryotic organisms is basically the same, with the following differences:

1. Chromosome structure. A prokaryotic chromosome is circular. Eukaryotic chromosomes are linear with ends called telomeres. 2. Origins of replications. A prokaryotic chromosome has one unique origin of replication. Eukaryotes have multiple origins to accommodate the much larger size of their chromosomes

DNA replication components and steps

1. Initiator Protein: recognizes where to begin 2. Helicase Enzyme: Lands at the origin- starts breaking the Hydrogen bonds between the bases, *working in both directions, at the replication fork 3. SSBP's (Single Stranded Binding Proteins)-keep the strands from joining again 4. Primase Enzyme: the RNA "primer" marker on the open end- marks where to begin adding nucleotides 5. DNA Polymerase: adds complimentary nucleotides, one at a time (A to T, G to C)

Draw the experiment of Alfred Hershey & Martha Chase (1952)

1. Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. 2. Agitate in a blender to separate phages outside the bacteria from the cells and their contents. 3. Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. 4. Measure the radioactivity in the pellet and the liquid. Batch 1: Phages grown with radioactive sulfur (32s) Batch 2: Phages grown with radioactive phosphorus (32p) Results: The experiment showed that T2 proteins remain outside the host cell during infection, while T2 DNA enters the cell. • Methionine (Met, M) - "start" amino acid, very hydrophobic

How the Neurospora crassa experiment worked

1. Mutated conidia 2. crossed with wild type of the opposite mating types 3. microscopic ascospores dissected and transfered one by one to culture tubes 4.Hundreds of tubes of complete medium inoculated with single ascospores 5.Conidia (asexualspores) from each culture then tested on minimal medium 6. Conidia from the cultures that fail to grow on minimal medium indicates a nutritional mutant, it is then tested on a variety of suplemented media. These suplemments are the 20 amino amino acids that range from glycine to cystine. The experiment has: • one minimal (control) • one minimal + amino acids (the nutrional mutant) • one minimal with vitamins • one complete Conclusion: the one amino acid that cause the minimal cell to thrive was...

RNA Capping

1. Post-transcriptional 2. Involves adding a 7me Guanosine nt to the first (RNA) nt in an unusual way, and often methylation of the first few nt of the RNA.

Functions of the PolyA-Tail

1. Promotes mRNA stability - De-adenylation (tail shortening) can trigger rapid degradation of the RNA 2. Enhances translation - promotes recruitment by ribosomes - bound by a polyA-binding protein in the cytoplasm, - synergistic stimulation with Cap!

Capping function

1. Protection from some ribonucleases 2. Enhanced translation* 3. Enhanced transport from nucleus 4. Enhanced splicing of first intron for some pre-mRNAs *Also functions of the polyA-tail

The processing to convert the pre-tRNA to a mature tRNA involves five steps.

1. The 5' end of the pre-tRNA, called the 5' leader sequence, is cleaved off. (edited) 2. The 3' end of the pre-tRNA is cleaved off. 3. In all eukaryote pre-tRNAs,, a CCA sequence of nucleotides is added to the 3' end of the pre-tRNA after the original 3' end is trimmed off. The CCA at the 3' end of the mature tRNA will be the site at which the tRNA's amino acid will be added. 4. Multiple nucleotides in the pre-tRNA are chemically modified. On average about 12 nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to pseudouridine (w), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D). But over 100 other modifications can occur. 5. Splicing « eukaryotic pre-IRNAs have introns that have to be spliced out, Introns are rarer In bacterial pre-RNAs, but do occur occasionally and are spliced out. (edited) 6. Charging - Attaching this amino acid is called charging the tRNA. In eukaryotes, the mature tRNA is generated in the nucleus, and then exported to the cytoplasm for charging.

Multiple Steps in Gene Expression after Transcription

1. Transcription 2. 5' Capping 3. 3' maturation: cleavage & polyadenylation 4. Splicing 5. Transport of RNA to Cytoplasm 6. Stabilization/Destabilization of mRNA

The equation of the reaction

1.Amino Acid + ATP -> Aminoacyl-AMP + PPi 2.Aminoacyl-AMP + tRNA - Aminoacyl-tRNA + AMP Altogether, the highly exergonic overall reaction is as follows: Amino Acid + tRNA + ATP -> Aminoacyl-tRNA + AMP + PPi

DNA REPLICATION BOOK STEPS

1.Helicase unwinds the DNA, producing a replication fork. Single-strand binding proteins prevent the sing strands of DNA from recombining. Topoisomerase removes twists and knots that form in the double-stranded template as a result of trhe unwinding induced by helicase 2.Primates initiates DNA replication at special nuecleotide sequences called origins of replication with short segments of RNA nucleotides called RNA primates (marker) 3.DNA polymerase attaches to the RNA primers and begins elongation, the adding of DNA nucelotides to the complementary strand 4. The leading complemntary strand is assembled contunously towards the replication fork as the double helix DNA uncoils 5.The lagging complementary strand is assembled away from the replication fork in multiple, short Okazaki fragments. Each new Okazaki fragments begins when DNA polymerase attaches to an RNA primer 6. The Okazaki fragments are joined by DNA ligase 7.The RNA primers are replaced with DNA nucelotides Energy for elongation is provided by two aditional phsphates that are attached to each new nucleotide, make a total of 3 phosphates attached to the nitrogen base. Breaking bonds holding the two extra phosphates provides chemical energy for this process

Translation termination

1.stop codon (UAG, UGA, UAA) enters the ribosome A-site 2.Recongition of the stop codon by release factor eRF1 3.Hydrolysis of the P-site-bound peptidy -tRNA and release of the nascent polypeptide

The ribosome is composed of a large and small subunit

30S 50S 70S •The large and small subunits undergo association and dissociation during each cycle of translation

Initiation of translation in Eukaryotes

In eukaryotes, the initiation of translation involves several key steps. It begins with the small ribosomal subunit binding to the mRNA at the 5' cap, and the initiation factors facilitate the assembly of the pre-initiation complex. The mRNA is then scanned until the start codon (usually AUG) is identified. Regarding your mentioned terms: 1. **A site (aminoacyl site):** This is where the incoming aminoacyl-tRNA binds to the mRNA codon. The amino acid is then transferred to the growing polypeptide chain. 2. **P site (peptidyl site):** This is where the tRNA carrying the growing polypeptide chain is located. Peptide bond formation occurs between the amino acid on the tRNA in the P site and the newly arrived aminoacyl-tRNA in the A site. 3. **E site (exit site):** After the transfer of the growing polypeptide chain to the aminoacyl-tRNA in the A site, the spent tRNA is moved to the E site. The tRNA then exits the ribosome, making room for a new aminoacyl-tRNA to enter the A site. This orchestrated movement of tRNAs and synthesis of the polypeptide chain continue in the elongation phase of translation.

Four requirements for DNA to be genetic material

Must carry information • Cracking the genetic code Must replicate • DNA replication Must allow for information to change • Mutation Must control the expression of the phenotype • Gene function

Wobble pairing and degeneracy

Non-standard ("wobble") base-pairing between the first anticodon and the third codon bases In addition to wobble base pairing, the third base in the codon (in most of the cases) shows degeneracy.

Oswald Avery, Colin MacLeod, & Maclyn McCarty (1944):

Process: 1. Used S (harmful) strain and lysed them 2. Opened up the cells 3. Isolated (DNA, proteins and other materials SEPERATELY) 4. Mixed R bacteria with these different materials (Only those mixed with DNA were transformed into S bacteria) Testtubes: • S DNA+ R Bacteria • S Proteins+ R Bacteria • Other S cell parts(sugar/RNA) + R bacteria History: • 1944 Oswald Avery, Maclyn McCarty and Colin MacLeod, identify Griffith's transforming agent as DNA. Their discovery is greeted with skepticism, in part because many scientists still believe that DNA is too simple a molecule to be the genetic material. • CONCLUSION: The molecule that carries the heritable information is DNA.

Purines and Pyramidines and their base pairings

Pyramidines: Cytosine, uracil, and thymine are PYramidines. Purines: Guanine and Adenine • Purines only pair with Pyrimidines • 3 hydrogen bonds required to bond Guanine & Cytosine • 2 hydrogen bonds required to bond Adenine and Thymine

The 3 possible models of DNA replication

Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand. Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule). Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or "hybrids," of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.

Hershey-Chase Experiment(introduction) (1952)

At the time of these experiments, it was known that phages(viruses) infect bacteria.In the first experiment Alfred hershey and martha chase substtuted radioactive sulfur for the sulfur in amino acids of the phage proteins and mixed these phages with E. coli bacteria. After separating bacteria from the growing medium through a centrifuge, they found out that the culture media which wasnt the bacteria was radio active, which means that the phage proteins didnt enter the bacteria. The second part of the expriment they substituted phosphorous for phosphourous in the phage DNA. Following the same procedure, they found out that the bacteria which wasn't the growing media was radioactive, indicating that phage DNA has entered the bacteria. In a follow up experiment the researchers found out that infected radioactive bacteria released new phages that were also radioactive. Conclusion: Hershey and chase concluded that the radioactive Dna from the phages provided genetic information to make new ones in the bacteria

Three bases lingo

DNA: ATC mRNA:UAG Anti-codon:AUC

A peptide bond is a

Dehydration synthesis

DNA

Deoxyribonucleic acid • Made up of subunits called nucleotides • Nucleotide made of: 1. Phosphate group 2. 5-carbon sugar 3. Nitrogenous base

What did Griffith discover in detail (introduction) ?

Genetic information can be transferred from dead bacteria to living ones. Microbiologist Frederick Griffith experimented with 2 strains of bacteria. On e which produces a polsaccharide coat that causes pneuomonia, and a mutant one without the coat which doesnt cause pneumonia in mice. Griffith killed the disease-causing bacteria and this displated that they no longer cause pneuomonia. He then injected both the dead strain and live bacteria that doesnt cause the disease. These mice died, griffith found live bacteria in mice that had polysaccharide coats. The descendants of these bacteria also had polysaccharide coats and would cause disease. Conclusion: Griffith conluded genetic information from dead bacteria with polysaccharide coats transfromed the bacteria without the cots giving them the ability to make coats and to cause disease. This is called transformation which is known as the ability to absrove genetic information and express it

Rules of Wobble Hypothesis

I. The first two bases of the codon and the last two bases of the anticodon undergo normal Watson-Crick base pairing. That is, hydrogen bonds are formed between adenine (A) and uridine (U), guanine (G) and cytosine (C), only. II. Less stringent rules of base pairing apply at the remaining position and non-Watson-Crick base pairing may take place. The bases that undergo such pairing are also referred to as wobble base pairs. This allows the anticodon of a single form (amino acid specific) ostRNA to pair with more than one codon in the mRNA.

What do Avery, Macleod and McCarty do (introduction) ?

Ideitified DNA as the heridtary information of a cell. Using the same bacteria from the Griffith experiment, Oswald Avery and his mates removed proteins and polysaccharides from the dead, pneomonia causing bacteria. They found that remaining material was still able to transform bacteria, giving harmless bacteria the ability to cause disease. Further test confirmed that it wasnt RNA but a substance with the same properties as DNA

Polyadenylation (or Poly(A)) signal, site and tail

The polyadenylation or Poly(A) is the process required for the synthesis of messenger RNA (mRNA) in which an endonucleolityc RNA cleavage is coupled with synthesis of polyadenosine monophosphate (adenine base) on the newly formed 3' end. The sequence elements for polyadenylation include the polyadenylation signal (POLY A_SIGNAL) and the polyadenylation site (POLYA_SITE). In mRNA or cDNA the added stretch of polyadenosine monophosphate is the polyadenylation tail (POLYA_TAIL). The polyadenylation signals are located downstream of the 3' exons. The polyadenylation site is the cleavage site at which POLYA_ TAIL is added in mRNA. localized downstream of the POLYA_SIGNAL. The sequence at/or immediately 5' to the site of RNA cleavage is frequently (but not always) CA. A 'G-U-rich' element or Downstream element usually lies just downstream of the POLYA SITE, which is important for efficient processing Poly(A) tail: Stretch of adenosine monophosphate (with only adenine bases) at the 3' end of mRNA or cDNA Polyadenylation site

vocab

Topoisomerases- Prevents torsion by DNA breaks Helicases- separates 2 strands Primase- RNA primer synthesis Single strand binding proteins- prevent the process of reformation of a double-stranded DNA molecule from single strands DNA polymerase- synthesis of new strand Tethering protein- - stabilises polymerase DNA ligase- It brings all the molecules of the newly formed DNA or RNA strands together and permanently binds them with a phosphodiester bond, so that they dont just fall apart

Transcription process

Transcription- process that makes mRNA from DNA 1. DNA unzips into 2 separate strands A. DNA Helicase is the enzyme that breaks H-bond 2. Free floating RNA NITROGEN BASES in the nucleus pair up w/unzipped DNA NITROGEN BASES(RNA Polymerase): A. Cytosine(C) pairs with Guanine (G) -(G) with (C) B. Uracil(U) pairs with Adenine(A) -(A) with (U) C. Thymine (T) pairs with Adenine (A) *** remember (T) is only with DNA 3. After all the pairing is done: • a single strand of RNA has been produced. 4. Genetic code from DNA is transferred to mRNA 5. The code obtained from, DNA lets the mRNA know which amino acids to pick up: • code is a set of 3 nitrogen bases = Codon

Watson, Crick, Wilkins, and Franklin determining the structure of DNA (introduction(

Using DNA prepared in the lab of Maurice Wilkins, Rosalind produced an x-ray diffraction of DNa known as photo 51. Image diffraction creates black and white pattern of spots that reveal structural characteristics of crystals. Fir DNA, the pattern revealed two strands wrapping around eachother. Rosalind also proposed that sugar-phosphate material formed the outside of thje double helix because of its hydrophilic properties, while hydrophobic nitrogenous bases were located on the inside. Conclusion: Using that infromation, James Watson and Francis Crick proposed a model of DNa resemblin a double helix, where the vertical sides are sugar-phosphate and the runfs are pair of nitrogen bases in which adenine pairs with thymine and guanine pairs with cytosine.

You are studying a biochemical pathway and isolate mutants I, ll, and III. Mutant I can grow if you supplement the medium with z. Mutant II can grow if you supplement the medium with X, Y, or Z. Mutant Ill can grow if you supplement the medium with X and Z, but not if you supplement the medium with Y. What is the order of X, Y, and Z in this biochemical pathway? make a table and pathway

X Y Z mut 1: - - + mut 2: + + + mut 3: + - + precursor--(2)-->(Y)---(3)---->(X)---(1)-->(Z)

formation of phosphodiester bond

condensation reaction between two nuclei ain vb

The start codon on mRNA always codes for

methionine

TRANSFER RNA

tRNA molecules match amino acids to codons in mRNA

Eukaryotes and the 5' cap specifcally

the 5' cap (cap-0), found on the 5' end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5' to 5' triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. It is referred to as a 7-methylguanylate cap, abbreviated m7G.

Promoters, transcription start site, and transcription unit

• "upstream" from the coding region of the gene. The promoter bases are NOT transcribed. • Once transcription begins, many RNA polymerases may read the DNA at once to get hundreds of mRNA templates made simultaneously -very efficient! • Promoter: DNA sequence where RNA polymerase binds to transcribe the gene • Transcription start site: the nucleotide where RNA pol initiates transcription • Transcription unit: the transcribed DNA

What is advantage of such degeneracy?

• - Point mutations are absorbed. •In normal situations anti codons match codons base by base. Occasionally point mutations change the bases, if the bases change the meaning of a codon for an amino acid also changes, then such mutations is called missense mutations. • If the base changes make amino acid coding combinations into nonsense codons where the codons don't recognize any aa.tRNA. There are no aa-tRNAs with anticodons, which can recognize such codons. Such mutations are called nonsense mutations. • Missense and nonsense mutations are often suppressed by mutations in respective tRNAs; such mutations are called suppressor mutations or intergenic suppressors.

DNA polymerse 1

• 1000 bases/second gives a lots of typos • 20 bases/second • proofreads & corrects typos • repairs mismatched bases • removes abnormal bases • repairs damage throughout life • reduces error rate from1 in 10,000 to1 in 100 million bases

DNA polymerase III

• 1000 bases/second! • main DNA builder

Alfred Hershey and Martha chase

• 1952: Alfred Hershey and Martha Chase (Text-p 262) Demonstrated that DNA, not protein, is involved in viral reproduction. The "transforming agent" • Tagged the protein coat of one bacteriophage (virus that infects bacteria) with one radioactive isotope, and the viral DNA of a second sample with a different isotope (red) • Infected, Blended, centrifuged: protein still outside the cell, DNA in the portion with the bacterial cells

History of james watson and francis, crick along with maurice wilkins

• 1953 James Watson and Francis Crick discover the molecular structure of DNA. Compiled data/made model "The forest through the trees" •1962 Francis Crick, James Watson, and Maurice Wilkins receive the Nobel Prize for determining the molecular structure of DNA.

The anti parralel structure of DNA

• 5' end: Phosphate group • 3' end: OH group • Antiparallel • Hydrogen Bonding between-individually weak, collectively strong • Built in a 5' to 3' direction, Read 3' to 5'

tRNA Contains Modified Bases

• 81 examples of modified bases in tRNAs have been reported. • Modification usually involves direct alteration of the primary bases in tRNA, but there are some exceptions in which a base is removed and replaced by another base.

tRNAs are Processed from Longer Precursors

• A mature tRNA is generated by processing a precursor. • The 5' end is generated by cleavage by the endonuclease RNAase P. • The 3' end is generated by multiple endonucleolytic and exonucleolytic cleavages, followed by addition of the common terminal trinucleotide CCA.

In what direction is DNA synthesized?

• Adding bases • can only add nucleotides to 3' end of a growing DNA strand • need a "starter" nucleotide fo bond to •strand only grows 5' to 3' direction

Cracking the Code

• All 64 codons were deciphered by the mid-1960s •Of the 64 triplets, 61 code for amino acids; 3 triplets are "stop" signals to end translation • The genetic code is redundant (more than one codon may specify a particular amino acid) but not ambiguous; no codon specifies more than one amino acid • Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced

tRNAs Are Charged with Amino Acids by Aminoacyl-tRNA Synthetases

• Aminoacyl-tRNA synthetases are a family of enzymes that attach amino acid to tRNA, generating aminoacyl-tRNA in a two-step reaction that uses energy from ATP. • Each tRNA synthetase aminoacylates all the tRNAs in an isoaccepting (or cognate) group, representing a particular amino acid.

Elongation of the RNA Strand

• As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time • Transcription progresses at a rate of 40 nucleotides per second in eukaryotes • A gene can be transcribed simultaneously by several RNA polymerases • Nucleotides are added to the 3' end of the growing RNA molecule

DNA Replication

• Begins at Origins of Replication • Two strands open forming Replication Forks (Y-shaped region) • New strands grow at the forks • As the 2 DNA strands open at the origin, Replication Bubbles form

Replication: 2nd step

• Build daughter DNA strand • add new complementary bases • DNA polymerase III

Ribozymes

• Catalytic RNAs molecules that function as enzymes; involved in splicing • Non-protein biological catalyst (telomerase, and splicesomes)

What Are Mutations?

• Changes in the nucleotide sequence of DNA • May occur in somatic cells (aren't passed to offspring)

Draw a condensation reaction between two nucleotides

• Condensation reaction between two nucelotides. The OH group of the phosphate group combines with the OH from the dexirbose sugar on the 3'. Resulting in a Oxygen linked to a phosphate group. The H from the OH group that leaves combines with the OH group making one water molecule. Result:OH+H= H2O and a phosphodiasther bond between two nucleotides

Basic Principles of Gene Expression

• DNA encodes hereditary information (genotype) -> decoded into RNA -> protein (phenotype) • DNA-----(Transcription)----->RNA--------(Translation)-->Protein

DNA vs RNA

• Double Helix vs Single strand • DNA- deoxyribose sugar (one less O than ribose) • RNA- Ribose sugar • 4 Nitrogen Bases DNA: -A-T -C-G RNA: -A-U -C-G • DNA stays in the nucleus, RNA made in the nucleus, works in the cytoplasm... where? • DNA contains information to direct development; RNA: 3 main types carry out DNA directions: -mRNA = messenger -tRNA = transfer -rRNA = ribosomal

Telomere

• End of a linear chromosome • Repetetive(TTAGGG)

increased complexity of genes splicing with the proein "Fibrillin"

• Fibrillin is a protein that is part of connective tissue. Mutations in it are associated with Marfan Syndrome • Syndromes: (long limbs, crowned teeth elastic joints, heart problems and spinal column deformities. The protein is 3500 aa, and the gene is 110 kb long made up of 65 introns. • Titin has 175 introns. • With these large complex genes it is difficult to identify all of the exons and introns.

Synthesis of an RNA Transcript The three stages of transcription(Draw it)

• Initiation • Elongation • Termination

Telomerase

• Key to immortatlity • An ezyme with RNA and protein components • Adds telomere repated directly to 3' overhand (uses its own RNA AS A TEMPLATE) • vERTEBRATE REPEAT dna on 3' end TTAGG • Telomerase RNA template AAUCCC

3 Types of RNA:

• Messenger RNA: (mRNA) carries nucleotide sequence from nucleus to ribosome • Transfer RNA: (tRNA) picks up amino acid in cytoplasm and carries them to ribosome • Ribosomal RNA: (rRNA) found in ribosome, joins mRNA and tRNA; forms protein

Are Mutations Helpful or Harmful?

• Mutations happen regularly • Almost all mutations are neutral • Chemicals & UV radiation cause mutations • Many mutations are repaired by enzymes • Some type of skin cancers and leukemia result from somatic mutations

Opening and closing the nucleus

• Next, the ends of the mRNA must be modified to signal to the pore to "open" and "close" • A cap is added 5' end • A poly A tail is also added to the 3' end • Remember synthesis is 5'-3' so the 3' end is always the tail!

Before mRNA can leave the nucleus, it must be modified, expression and exons, and introns

• Not all of the DNA is expressed at once. • Usually only one gene or a few genes at a time •Exons are the part of the mRNA transcript that are EXPRESSED • Introns are the INERT part

Maurice Wilkins (1952)

• Photographed DNA using x-ray crystallography • Worked with another scientists named Rosalind Franklin • Awarded the 1962 Nobel Prize for Physiology or Medicine with Watson and Crick

Vocab of introns exons etc

• Primary mRNA = exons + introns • INTRONS = discarded, noncoding region of mRNA •EXONS = the coding regions, spliced together RNA are called also = Mature mRNA

Transcription- mRNA

• Process of copying DNA into mRNA: • Every 3 bases will code for 1 amino acid.....building a protein

How many replication bubbles do prokaryotes and eukaryotes have

• Prokaryotes (bacteria) have a single bubble • Eukaryotic chromosomes have MANY bubbles

RNA Polomerase Binding and Initiation of Transcription

• Promoters signal the transcriptional start point and usually extend several dozen nucleotide pairs upstream of the start point • Transcription factors mediate the binding of RNA polymerase and the initiation of transcription • The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex • A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

Deoxyribose nucleis acid type of nucleic ac

• RNA • DNA function: -To hold genetic code -Genetic code = genetic instructions to make proteins • DNA is found in nucleus of eukaryotic cells • Found in nucleoid region in prokaryotm

RNA

• RNA: ribonucleic acid • Carries out protein synthesis • Differences from DNA: • different sugar (ribose) • single strand • different base - no thymine - URACIL instead

Why do telomeres get shorter?

• Since RNA polymerase is so big and takes so much space, it sits on the end of DNA and replicates it • However, the place it sits on doesnt get replicated causing it to become shorter and shorter. This is why telomeres exists.

Functional and Evolutionary Importance of Introns

• Some genes can encode more than one kind of polypeptide •Different combinations of exons can be spliced togetherand this is called alternative RNA splicing • Increases the potential number of different proteins (and thus functions) in an organism which Increases adaptive potential

Telomerase end telomere length in aging

• Telomerase stops slowly at embryonic stage, thats why telomeres get shorter as you age

promoter and transctiption unit

• The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the • The stretch of DNA that is transcribed is called a transcription unit

Termination of Transcription

• The mechanisms of termination are different in bacteria and eukaryotes • In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification • In eukaryotes, RNA polymerase Il transcribes the polyadenylation signal sequence; the RNA transcript is released 10-35 nucleotides past this polyadenylation sequence

Pathway can be deduced using the following logic:

• The mutant strain is blocked farther along in a pathway if fewer intermediate compounds permit the strain to grow OR • The mutant strain is blocked at earlier steps in the pathway if a larger number of intermediates enable the strain to grow.

Energy of Replication

• The nucleotides arrive as nucleosides • DNA bases with P_P_P •P-P-P = energy for bonding •Energy for elongation is provided by two aditional phsphates that are attached to each new nucleotide, make a total of 3 phosphates attached to the nitrogen base. Breaking bonds holding the two extra phosphates provides chemical energy for this process •ATP-ADP-AMP •GTP-GMP •TTP-TMP •СТР-CMP

leaving the nucleus

• The nucleus has pores formed by proteins • DNA is double stranded, too fat to fit through the pore! • mRNA is a single strand and once it is properly modified it can exit through the pore so translation can occur at a ribosome

Codon-Anticodon Recognition Involves Wobbling

• The pairing between the first base of the anticodon and the third base of the codon can vary from standard Watson-Crick base pairing according to specific wobble rules.

Teromerase and telomere length in tumors

• Tumor cells have shorter telomeres • However telomerase is reactivated causing the cell to divide indefinetely because telomeres are replaced

Replication: 1st step

• Unwind DNA done by helicase enzyme • Unwinds part of DNA helix • stabilized by single-stranded binding proteins

Fredrick Griffith (1928) process

• Used the Pneumococcus bacteria Include 2 types: • a virulent S strain with a Smooth coat -kills mice • a non-virulent R Rough strain -does not kill mice. • Heat destroys (kills) living cells!!! • When heated Smooth (harmful) cells (DEAD) are mixed with living Rough (benign) cells and injected into mice, the mouse dies.

Freidrich Miescher (1868):

• discovered DNA • Isolated something new from the nuclei of eukaryotic cells • Later called DNA!!!

Gregor Mendel (1866):

• discovered that inherited traits are determined by discrete units, or 'genes,' - passed on from the parents.

Telomere structure

•3' overhang on G strand. The end js bound to an end-specific telomere protein •Loops back on itself •feature distinct structures such as the D-loop and T-loop. The D-loop, formed during telomere replication, involves the 3' overhang of the G-strand invading the double-stranded region. •Concurrently, the T-loop arises as the single-stranded G-rich overhang folds back on itself, stabilized by telomere-binding proteins. •This 3' overhang, specifically on the G-strand, prevents loss of genetic material during DNA replication. •Telomere proteins bind to these ends, safeguarding structural integrity. The T-loop acts as a shield, warding off degradation and fusion, thereby maintaining genomic stability. This intricate architecture plays a pivotal role in preserving telomeric DNA, crucial for cellular longevity and genomic health.

1928 Fredrick Griffith history

•A British medical officer, discovers that genetic information can be transferred from heat-killed bacteria cells to live ones. •This is called transformation, provides the first evidence that the genetic material is a heat-stable chemical. Griffith was trying to develop a vaccine against pneumonia when he discovered the phenomenon of transformation. •Scientists hypothesized that a chemical substance was transferred from the dead bacteria to the living cells and caused transformation

tRNAs Are Charged with Amino Acids by Aminoacyl-tRNA Synthetases#2

•Aminoacyl-tRNA synthetases catalyze attachment of amino acids to specific tRNAs •Each aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural features of its corresponding tRNA • Recognition of tRNA by tRNA synthetases is based on a particular set of nucleotides, the tRNA "identity set. » that often are concentrated in the acceptor stem and anticodon loop regions of the molecule.

Why and how does splicing work?

•An enzyme cuts out the intron and splices the exons together •This saves valuable ENERGY for the cell so it is not making proteins it doesn't need.

Eukaryotic and prokaryoticpre-mRNA processing

•Bacteria mRNA are functionally active as transcribed •Eukaeyotic pre-mRNA must be extensively processed to attant their functional forms •The modifcation that occurs at the 5' end of the primary transcript is called the 5' cap (m7 Gppp). In this modification. a 7-methylguanilate residue is attached to the first nucelotide of the pre mRNA by a 5'-5' linkage. •The 2' hydroxyl groups of the ribose residues of the first 2 nucelotides may also be methylated. •The 5' is important for transport of mRNA tp the cytoplasm, protection against nuclease degredation, and initiation of translation.

Dna primase, primer and polymerase

•Before new DNA strands can form, there must be RNA primers present to start the addition of new nucleotides •Primase is the enzyme that synthesizes the RNA Primer •DNA polymerase can then add the new nucleotides

Alfred Hershey & Martha Chase (1952)

•Confirmed DNA was genetic material •Used bacteriophages (viruses) •HYPOTHESIZED -> DNA, not protein, is the hereditary material

Erwin Chargaff (1950)

•Discovered a 1:1 ratio of adenine to thymine and guanine to cytosine in DNA samples from a variety of organisms.

1942 - George Beadle & Edward Tatum and the one gene one enzyme hyptehsis

•Discovered that genes act by regulating definite chemical events. •Studied relationships between genes and enzymes in the haploid fungus Neurospora crassa (orange bread mold). •One Gene-One Enzyme Hypothesis: Each gene controls synthesis/activity of a single enzyme. -"one gene-one polypeptide"

Eukaryotic cells modify RNA after transcription

•Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm •During RNA processing, both ends of the primary transcript are usually altered • Also, usually some interior parts of the molecule are cut out, and the other parts spliced together

Beadle and Tatum proposed: "One Gene-One Enzyme Hypothesis" however...

•However, it quickly became apparent that... •More than one gene can control each step in a pathway (enzymes can be composed of two or more polypeptide chains, each coded by a separate gene) .•Many biochemical pathways are branched .•"One Gene-One Enzyme Hypothesis" became "One Gene-One Polypeptide Hypothesis"

what is marfan's syndrome, and how is it inherited?

•Marfans Syndrome is a disorder that affects the connective tissue, so its not as strong as it would be. This results in long limbs. •This is passed down by a non sex-linked (autosomal) dominant pattern, which means one copy of the altered gene in each cell is plentiful to cause the disorder. At least 25 percent of Marfan syndrome cases result from a new alteration in a gene.

Modified Bases Affect Anticodon-Codon Pairing

•Modifications in the anticodon affect the pattern of wobble pairing and therefore are important in determining tRNA specifichy

Life cycle of the haploid fungus Neurospora crassa (orange bread mold):

•Neurospora reproduces in a web-like pattern with asexual spores called conidia, which are orange in color. •Neurospora is haploid, so mutations are spotted easily (unlike diploid organisms). •Life cycle is short, and grows on simple media (N, C, biotin). • Sythesizes all other substances from these basic nutrients. •Propagates vegetatively (asexually) by dispersal of mycelium and asexual spores (conidia), OR •Sexually, by means of two mating types, A and a (only A will mate with a and result in a gamete fusion and meiosis. • Meiosis leads to 4 haploid nuclei (2A, 2a) • After one round of mitosis, 8 ascospores (4A, 4a).

Rosalind Franklin (1952)

•Obtained sharp X-ray diffraction photographs of DNA (Photo 51) •Watson and Crick used her data - revealed its helical shape •Watson and Crick went on to win Nobel Prize (1962) for their DNA model

Fredrick Griffith (1928)

•Studied effects of virulent (disease-causing) bacteria vs. nonvirulent bacteria injected into mice Used transformation: • Inserted foreign DNA and changed protein/ trait • believed that the transforming agent was an inheritance molecule •R strain is benign (Lacking a protectivo capsule, it is recognized and destroyed by host's immune system) •S strain is virulent (Polysaccharide capsule prevents detection by host's immune system)

Properties of the genetic code

•The genetic code is composed of nucleotide triplets. • The genetic code is nonoverlapping. •The genetic code is comma-free. •The genetic code is degenerale. (yes) •The genetic code is ordered. (5 to 3) • The genetic code contains start and stop codons. (yes) •The genelic code is nearly universal,

Evolution of the Genetic Code

•The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals •Genes can be transcribed and translated affer being transplanted from one species lo another

Problems with cracking the code

•To match nucleolide-by-nucleolide, employing complementary base pairing. there should be 61 different tRNA with different anti codon nucleotide sequences, but the total number of RNAs found in any system is less than 61 and in most case it is 22 to 31 maximum. If that is the case how on earth the tRNAs can match 61 codons. This paradox is solved by what is famously called wobble base pairing proposed by F.C. Crick. In this, the anticodon 5' end base in tRNA has the ability to pair with more than one base found at the 3rd base at 3' end of the codon (mRNA). Orientation of codons and anti codons is anti parallel. The third base in the codon base pairs with the first base of the anti codon.

A P E sites

•Translation initiation factors hold eukaryotic mRNAs in circles •Each mRNA can be translated simultaneously by multiple ribosomes • A site - Amino acid is dropped off • P site - Polypeptide builds • E site - tRNA exits (after giving its amino acid to building polypeptide chain)

What is RNA splicing carried out by and what happens during it?

•carried out by splicedsomes Spliceosomes •complex of proteins and several small nuclear ribonucleoproteins (snRNEs) •spliceosomes recognize the splice sites (specific BNA sequences) cleave out introns and splice together exons (coding region)

NHEJ

•chops pieces off and glues some back on which leads to mutations and that leads to cancer •This is why telomeres are hidden •Non-Homologous End Joining (NHEJ) is a DNA repair mechanism employed by cells to fix double-strand breaks in DNA. NHEJ works by chopping off damaged DNA ends and then rejoining the broken ends, often without using a template. This process is error-prone and can introduce mutations, as the repair machinery may not precisely align the ends or may add or delete nucleotides during the rejoining process. •Telomeres, being the protective caps at the ends of chromosomes, are particularly sensitive to these repair mechanisms. If exposed, the natural ends of linear chromosomes might be recognized as double-strand breaks, triggering NHEJ and potentially leading to errors and mutations. To avoid this, telomeres hide their natural ends by forming protective structures like the T-loop. This helps prevent unnecessary DNA repair activities at the chromosome ends, reducing the risk of mutations that could contribute to conditions such as cancer. The intricacies of telomere protection highlight the cell's commitment to maintaining genomic stability.

The central dogma of biology

The central dogma of molecular biology outlines the flow of genetic information within a biological system. The central dogma consists of three main processes: 1. **Replication:** The genetic information encoded in DNA is duplicated during cell division. This ensures that each daughter cell receives a complete set of genetic instructions. 2. **Transcription:** The information in DNA is transcribed into messenger RNA (mRNA) in the nucleus. This process involves the synthesis of an RNA molecule that carries the genetic code from DNA to the ribosomes in the cytoplasm. 3. **Translation:** The information carried by mRNA is then translated into proteins at the ribosomes. Transfer RNA (tRNA) molecules bring amino acids to the ribosomes, where they are linked together according to the sequence specified by the mRNA. This unidirectional flow of genetic information—DNA to RNA to protein—constitutes the central dogma.

Draw the replication bubble

During DNA replication, a replication bubble forms as a result of the unwinding and separation of the DNA double helix. Initiated at specific sites called origins of replication, helicase enzymes unwind the DNA strands, creating a Y-shaped replication fork. The region between the separated DNA strands constitutes the replication bubble, where DNA polymerases can access the single-stranded templates and synthesize new DNA strands. This process occurs bidirectionally, with replication proceeding in both directions away from the origin. The synthesis of new DNA strands within the replication bubble ensures the semiconservative replication of DNA, where each newly formed DNA molecule comprises one parental strand and one newly synthesized strand. As the replication process continues, multiple bubbles may form along the DNA molecule, allowing for the efficient and simultaneous replication of the entire DNA during cell division.

Dna replication

During interphase of the cell cycle a second chromatid copy of DNA is made, it invoolves separating the doubl-stranded DNA molecule into two strands, each of which serves serves as a template to make new complentary strands. The result is two completely identical double stranded molecules of DNa.1

Elongation of proteins

During the elongation phase of translation in eukaryotes: 1. **Codon Recognition:** Aminoacyl-tRNA binds to the A site, recognizing the mRNA codon through base-pairing. 2. **Peptide Bond Formation:** Peptide bond formation occurs between the amino acid on the tRNA in the A site and the growing polypeptide chain on the tRNA in the P site. This step is catalyzed by peptidyl transferase activity within the large ribosomal subunit. 3. **Translocation:** The ribosome advances along the mRNA, moving the tRNA in the A site, which now holds the growing polypeptide chain, to the P site. The empty tRNA in the P site is moved to the E site and then released from the ribosome. 4. **A Site Readiness:** The mRNA is read three nucleotides at a time (codons), and the process repeats as the next aminoacyl-tRNA enters the now vacant A site. This cyclic process continues, synthesizing the polypeptide chain in the order dictated by the mRNA codons until a stop codon is reached, marking the end of translation.

semiconservative replication

Each of new double stranded DNA molecule consists of a single strand of old DNA which was a template and a single strand of new replicated DNA which is the complimentary strand

DNA replication process

Enzyme helicase unwinds DNA, forming Y-shaped replication fork.Single stranded binding proteins attach to each strand of uncoiled DNA to keep them seperated. As helicase unwinds the DNA, it forces the double helix int he front to twist. A group of enzymes known as topoisomerases break and rejoin the double helix allowing twists to unravel and prevent knots from happening Since DNA is made of two opposing DNA strands, The uncoiled DNA consists of (template strand 3'-5'). The enzyme that assembles the new DNA, which is the DNA polymerase, moves in the 3'-5' direction along each template strand. A new (complimentary) strand grows in the 5'-3' antiparralel direction. For the 4'-5' template strand, replication occurs continuesly as the DNA polymerase follows the replication fork, assembling a 5'-3' complentary strand. This complimentary strand is called the leading strand For the 5'-3' template strand, DNA polymerase moves away from the uncoiling replication fork. This is because it can assemble nucelotides only as it travels in the 3'-5' direction. As the helix is uncoiled, DNA polymerase assembles short nuceotide segments along the template strand in the direction awat from the replication fork. After each compliment strand is assembled, the DNA polyemrase must return back to the replication fork to begin assembling the next segment. These short fragment of complintary DNA are called okazaki fragments. The okazaki fragments are connected by the DNA ligase, producing a single complimentary strand. Because theis complimentary strand requires more time to assemble than the leading strand, it is known as the lagging strand DNA polymerase is able to attach nucleotides only to an already existing complemnatary strand. Therefore, to initiate a new compliemntaary strand, another enzyme known as primase begins replication with

Meselson-Stahl Experiment 1958 Disproves Conservative and dispersive

Observations: They began by growing E. coli in medium, "heavy" isotope of nitrogen containing heavy ‍15N , the bacterua used it to synthesize new DNA. Then, the bacteria were switched to medium containing a "light" ‍ 14N isotope and allowed to grow for several generations. DNA made after the switch would have to be made up of ‍ 14N because it is the only nitrogen available for DNA synthesis. Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. They then measured the density of the DNA using density gradient centrifugation. Density gradient centrifugation allows very small differences—like those between 15N- and ‍14N -labeled DNA—to be detected. Generation 0: The DNA isolated from cells at the start of the experiment showed a single band in the centrifuge, indicating that it contained only heavy 15N. Generation 1: After one round of DNA replication, the DNA showed an intermediate band, suggesting a hybrid molecule containing both heavy 15N and light 14N. This result was inconsistent with the conservative model but supported both dispersive and semi-conservative models. Generation 2: The second generation DNA produced two bands—one at the position of the intermediate band from the first generation (hybrid molecule) and another higher band (pure light 14N molecule). This result supported the semi-conservative model. Generations 3 and 4: Subsequent generations showed a pattern consistent with semi-conservative replication. The hybrid band became fainter, representing a smaller fraction of the total DNA, while the light band became stronger, representing a larger fraction.

Central Dogma in Eukaryotes and prokaryotes

The Central Dogma of molecular biology describes the flow of genetic information within a biological system. In both eukaryotes and prokaryotes, it consists of three main processes: 1. **Replication (Common to both):** The process by which DNA is copied to produce an identical DNA molecule. This occurs in the nucleus of eukaryotic cells and in the nucleoid region of prokaryotic cells. 2. **Transcription:** - **Eukaryotes:** Transcription takes place in the nucleus. The DNA is transcribed into a complementary RNA molecule (pre-mRNA). - **Prokaryotes:** Transcription occurs in the cytoplasm (no nucleus). The DNA is directly transcribed into mRNA. 3. **Translation:** - **Eukaryotes:** Translation occurs in the cytoplasm. The pre-mRNA is processed into mature mRNA, which carries the genetic code to the ribosomes. Ribosomes translate the mRNA into a polypeptide chain (protein). - **Prokaryotes:** Translation also occurs in the cytoplasm. mRNA is translated directly into a polypeptide chain by ribosomes. In summary, while the basic processes of replication, transcription, and translation are common to both eukaryotes and prokaryotes, there are differences in the cellular compartments where these processes take place and in the details of transcription and translation. Eukaryotes have a compartmentalized nucleus for transcription, whereas prokaryotes lack this membrane-bound organelle.

tRNAs structure and job and anti codons

The anticodon is the sequence of three nucleotides that base-pairs with a codon in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3' end of the tRNA. (RNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been synthesized.