Describe the roll of the following in DNA synthesis:

DNA polymerase III

Replicates DNA by matching base pairs.

Describe the roll of the following in DNA synthesis:

3' to 5' exonuclease

Proofreads new strads of DNA for errors

Describe the roll of the following in DNA synthesis:

RNase H

Removes the RNA primer

Describe the roll of the following in DNA synthesis:

DNA polymerase I

Bacterial enzyme that makes small stretches of DNA to fill in gaps between Okazaki fragments or during the repair of damaged DNA.

Describe the roll of the following in DNA synthesis:

Sliding Clamp

A protein fold that serves as a processivity-promoting factory in DNA replication.

Describe the roll of the following in DNA synthesis:

Clamp Loader

Loads sliding clamps onto the DNA template strands and disassembles the sliding clamp after replication has been completed.

Describe the roll of the following in DNA synthesis:

Helicase

Proteins that move along the phosphodiester backbone and separate two annealed strands of DNA.

Describe the roll of the following in DNA synthesis:

Gyrase

Enzyme that relieves stran while double-stranded DNA is being unwound (by helicase) by making breaks in DNA strands

Describe the roll of the following in DNA synthesis:

DNA ligase

An enzyme that repairs discontinuities in DNA molecules caused by the gyrase enzyme.

Describe the roll of the following in DNA synthesis:

Primase

Starting point for synthesis

Describe the roll of the following in DNA synthesis:

Single Stranded Binding Protein

Binds to single-stranded regions of DNA to prevent premature annealing, protect the DNA from being digested by nucleases, and remove secondary structures from the DNA to allow other enzymes to function upon it.

Describe the roll of the following in DNA synthesis:

DNA polymerase alpha

Initiates new strands.  Has primase and DNA polymerase activity.

Describe the roll of the following in DNA synthesis:

DNA polymerase delta

Base excision repair

Draw and label a replication bubble with 2 replication forks to show leading strands and 2 Okazaki fragments at each forks.

Show how the cell uses telomerase to prevent telomere shortening.

Telomerase is an RNA containing enzyme that uses RNA as a template for DNA synthesis to extend the non-shortened strand.  The RNA is complementary to telomere DNA (TTAGGG).

Diagram how the cell uses telomerase to prevent telomere shortening

As far as telomeres are concerned, why does size matter?

Short telomeres are characteristic of premature aging syndroms and age-related diseases.

Elizabeth blackburn and Carol Greider

1. Discovered the structure of and the function of telemorase in 1984.
2. Won the 2009 Nobel Prize in Physiology or Medicine.

Leonard Hayflick

Discovered that human cells divide for a limited number of times (Hayflick Limit).

Alexy Olovinkov

1. Noted that linear DNA cannot copy the telomere (end) of the DNA of the lagging strand (1973)
2. Suggested that telomere shortening may be related to aging (1973)

William Bateson

Discovered that PKU was inherited as a Mendelian recessive.

Jean Brachet

Recognized a correlation between RNA concentration and protein synthesis around 1940.

David Bonner

Characterized tryptophan requiring mutans and found that it was a likely precursor of arginine.

Herschel Mitchell

Demonstrated that one of the tryptophan requiring mutants was missing the enzyme (tryptophan synthase) that condenses indole and serine.

Marianne Grunberg-Monago

1. Discovered polynuclotide phosphorylase in 1995.
• That discovery lead to the synthesis of RNAs such as poly U, poly A, and poly C.

George Gamow

Defined, articulated, and attempted to solve the coding problem (DNA).

Robert Roeder

1. Discovered the 3 forms of RNA polymerase in eukaryotes (1969)
• Polymerase I: synthesizes rRNA
• Polymerase II: synthesizes most mRNA (protein coding genes)
• Polymerase III: primarily synthesizes tRNA and 5S RNA.

Roger Kornberg

1. Determined the structure of RNA polymerase II
2. Won the Nobel Prize in Chemistry (2006)

General Transcription Factor

1. Needed for transcription of all genes transcribed by a particular DNA polymerase
2. Generally involved in gene regulation

Specific Transcription Factor

Needed for transcription of particular (specific) genes under specific circumstances

Activator

1. Bind to the DNA and help recruit RNA polymerase and stabilize its binding to DNA.  
2. May recruit chromatin remodeling complexes.

Coactivator

Incrases transcription by relaxing the chromatin structure to allow greater access to a gene or by bringing in components of the basal transcription complex needed for transcription to occur.

Mediator

A protein complex that transmits the signal from transcription factors to the RNA polymerase in eukaryotic cells.

Alpha Amanatin

1. Deadly toxin synthesized by the mushrom Amanita phillodies.
• RNA polymerase I: resistant
• RNA polymerase II: highly sensitive
• RNA polymerase III: moderately sensitive

Actinomycin D

1. Class of polypeptide antibiotics isolated from soil bacteria (Streptomyces).
2. It was the first antibiotic isolated by Selman Waksman and H.B. Woodruff in 1940.
• RNA polymerase I: highly sensitive
• RNA polymerase II: slightly sensitive
• RNA polymerase III: slightly sensitive

Reverse Transcriptase

1. Discovered by David Baltimore and Howard Temin.
2. RNA can make DNA

Howard Temin and David Baltimore

1. Discovered reverse transcriptase
2. Won Nobel Prizes in 1975

Jerome P. Horwitz

Synthesized AZT in 1964 as an anticancer drug.

AZT

Developed as an anticancer drug (1964), but was found to be effective against AIDS (1985).

Joan Steitz

Discovered snRNPs

Outline, discuss, and state the significance and conclusions of the experiments of Adrian Srb and Norman Horowitz.

Srb and Horowitz grew arginine requiring mutans on intermediates.  They analyzed 3 classes of arginine requiring mutants.  By mating they found that the 3 classes were in 3 different genes.  The mutants grew on compounds structurally related to arginine.  They found that citrulline accumulated in the medium of those that could only grow on arginine.  Mutan class 2 accumulated ornithine.  Srb and Horowitz concluded that ornithine and citrulline were most likely precursors of arginine.

Outline, discuss, and state the significance and conclusions of the experiments of Archibald Garrod.

Garrod studied metabolic disorders such as alkaptonuria, which results in an accumulation of alkapton and its excretion in the urine.  Upon exposure to oxyge, the alkapton turns black.  Gerrod determined the metabolic steps leading to hemogentisic acid.  He realized that alkaptonurics cannot cleave the benzene ring.  Along with William Bateson, he discovered that the disease was inherited as a Mendelian recessive.  This was a significant discovery because it connected heredity (DNA) to metabolism (enzymes).

Outline, discuss, and state the significance and conclusions of the experiments of Heinz Frankel-Conrat.

Hienz Frankel-Conrat took 2 different types of viruses (type A and type B) and strupped off all the proteins, giving him proteins and RNA for both types of viruses.  He then performed a mix and match experiment where he added proteins from type A to the RNA of type B and proteins from type B to the RNA of type A.  He introduced these hybrid viruses to tobacco plants and examined the viruses that were produced by the plants.  The new viruses had type A RNA with type A proteinds and type B RNA with type B proteins.  This demonstrated that it was the nucleic acid that made the proteins.

Outline, discuss, and state the significance and conclusions of the experiments of Daniel Nathans, Norton Zinder, G. Notani, and J. Schwartz.

In 1962, Daniel Nathans, G. Notani, J. Schwartz, and Norton Zinder demonstrated that RNA could direct the synthesis of a protein.  They incubated an E. coli cell free, DNA free extract with ATP, labeled amino acids, and f2 phage RNA and obseved more labeled proteins with increasing amounts of f2 RNA.  They synthesized, labeled protein product and unlabeld phage coat proteins were analyzed by digesting with tripsin, followed by 2D electrophoresis.  9 peptides were found on the ninhydrin electropherogram and 8 peptides were found on the radioautogram.  Since they were the same, one can conclude that the synthesized product was probably a coat protein.

Outline, discuss, and state the significance and conclusions of the experiments of Sol Spiegelman and Benjamin Hall.

Spiegleman and Hall infected E. coli with T2 phage labeled with ³H DNA.  The T2 was allowed to infect E. coli in a ³²P medium.  Any RNA made in the medium was labeled as ³²P RNA.  The T2 ³H DNA was denatured.  The T2 ³²P RNA, T2 denatured ³H DNA,a nd the undenatured, unlabled T2 control DNA were separated by density gradient centrifugation.  The dentured T2 ³H DNA and the ³²P RNA formed hybrids among the separated strands (³²P RNA, T2 ³H DNA).  A new peak of ³²P was formed.  This peak was a T2 RNA/T2 DNA hybrid showing that the new T2 RNA synthesized after infection was complementary to T2 DNA. This shows that DNA is transcribed onto an RNA molecule before protein is synthesized.

Outline, discuss, and state the significance and conclusions of the experiments of Elliot Volkin and Lazarus Astrachan.

Elliot Volkin and Lazarus Astrachan used ¹⁴C bases and found that RNA labeled after T2 infection of E. Coli has a base ratio similar to that of T2 DNA rather that that of E. coli DNA.  This is exactly what one would expect if T2 DNA directs synthesis of T2 RNA, which is then translated into T2 protein.  They referred to the RNA as DNA-like RNA (dlRNA).

Outline, discuss, and state the significance and conclusions of the experiments of Francois Gros and Waclaw Szybalski.

Gros and Szybalski demonstrated that when E. coli galactose and lactose metabolizing genes are turned on, and RNA is synthesized that hybridizes to E. coli gal and lac DNAs.

Outline, discuss, and state the significance and conclusions of the experiments of Marshal Nirenberg and Heinrich Matthaei.

Nirenberg and Matthaei studied protein synthesis in cell free E. coli extracts.  The plan was to study the transfer of ¹⁴C amino acids to protein using various RNAs.  Matthaei noticed that poly A stimulated polypeptide synthesis when added to the system.  The thought was that it was binding nucleases allowing for more polypeptide synthesis.  It was assumed that poly A would be non-coding.  He ran a number of experiments and concluded that only polu U stimulates the synthesis of polyphenylalanine.  This means the code for phenylalanie is UUU.  The same experiments were performed in order to discover the codes for lysine (AAA) and proline (CCC).  Poly G cannot be used as a template because of internal pairing.

Outline, discuss, and state the significance and conclusions of the experiments of Marshal Nirenberg and Phillip Leder.

Mirenberg and Leder used trinucleotides to test for each amino acid assignment.  They incubated aa-tRNA, ribosomes, and trinucleotides on filters.  Only tRNA, ribosomes, or trinucleotides would remain on the filter.  They counted the number of items left on the filter to determine the nucleotide sequence of codons.

Outline, discuss, and state the significance and conclusions of the experiments of Severo Ochoa.

Ochoa synthesized amino acids using random polynucleotides (Poly U, Poly C, Poly UC, Poly UA, Poly CU).  It was discovered that combinations of the polynucleotides coded for the same amino acid.

Outline, discuss, and state the significance and conclusions of the experiments of Philip Sharp and Richard Roberts.

Sharp and Roberts hybridized EcoR1 restriction fragment of adenovirus DNA to the major capsid protein mRNA and found 3 loops of DNA.  They suggested that the DNA in the loops were spliced from the main body of the mRNA during the processing of a long precursor. 

Outline, discuss, and state the significance and conclusions of the experiments of Har Gobind Khorana.

Made DNA of defined sequence by chemical means and then made RNA from the DNA using RNA polymerase.  These RNAs were used extensively in experiments to decipher the genetic code.

How did the PaJaMa experiment lead to the development of the messenger hypothesis?

Jacob and Mond realized that an unstable molecule was responsible for turning on/off β-galactosidase synthesis when the repressor molecule dissociates/binds from/to the operator. 

State the function of the distal promoter.

State the function of the CAAT box.

A sequence often found in the upstream region of eukaryotic promoters and which binds transcription factors.

State the function of the GC box(es).

A sequence of contiguous nucleotides (GGGCG) along a DNA or RNA strand.

State the function of the proximal promoter.

1. Consists of 1 or more of the following upstream promoter elements:
• GC box(es)
• C_pG islands 
• CAAT boxes
• Response elements

State the function of the core promoter.

1. Consists of one or more of the following:
• The initiator box
• The TATA box
• TFIIB recognition element (BRE)
• The downstream initiator element (Inr)
• The downstream promoter elementt (DPE)

State the function of 5' and 3'.

RNA is made in a 5' to 3' direction.  5' is the starting point for transcription while 3' is the ending point.

State the function of the TATA box.

1. Binding site for a transcription factor that guides RNA polymerase II to the promoter in eukaryotes.
2. -26 to -32 nucleotides
3. TATA is the consensus sequence

State the function of the initiator sequence.

Three-base sequence on the messenger RNA that codes for the amino acid methionine and is the start command for synthesis.

-Code: AUG

State the function of the 5' UTR (5' untranslated region).

Regions of an mRNA between the 5' end and the translation start site.

State the function of the nucleotides +1, +100, -100.

1. Nucleotide +1: start of transcription
2. Nucleotide +100: 100 nucleotides after start of transcription
3. Nucleotide -100: 100 nucleotides before start of transcription.

State the function of the exons and intervening sequences.

1. Exon: a segment of a gene that codes for protein and that is still present in the mRNA after processing is complete.
2. Intervening sequence: segment of a gene that does not code for protein, but is transcribed and forms part of the primary transcript.
• also called introns

State the function of the translation start and translation stop.

1. Translation start: the mRNA codon (AUG) at which the ribosome initiates translation of the sequence into amino acids.
2. Translation stop: there are 3 codons (UAA, UAG, and UGA) that signal the end of protein synthesis

State the function of the enhancer and silencer.

1. Enhancers stimulate transcription
2. Silencers supress transcription

What role does the RNA polymerase carboxy terminus domain (CTD) (tail) play in transcription and RNA processing?

The CTD tail of RNA polymerase is responsible for recruiting the poly A addition enzymes.

Discuss the following mRNA modification that occurs during the maturation of mRNA:

CAPping

1. Refers to the addition of a special nucleotide at the 5' end by a special linkage.  Occurs as early as nucleotide #1, but no later than #20-40.  Occurs as transcription is taking place.
2. Serves 3 purposes:
• protects the mRNA from degradation
• stimulates transport of mRNA from nucleus to cytoplasm
• recruits small ribosomal subunit to increase the efficiency of translation
3. Discovered by Aaron Shatkin

Discuss the following mRNA modification that occurs during the maturation of mRNA:

Splicing: How does the cell know where to splice?

Discuss the following mRNA modification that occurs during the maturation of mRNA:

Splicing: What are snRNPs?

snRNPs are small nuclear ribonucleoproteins

Discuss the following mRNA modification that occurs during the maturation of mRNA:

Splicing: With real human examples, discuss splicing errors.

Splicing errors can occur if splice sites are skipped and/or if sequnces are mistaken for splice sites.  Up to 60% of known disease causing mutations affect splicing.  An example of a disease caused by splicing errors is Systemic Lupic Erythematosis.

Discuss the following mRNA modification that occurs during the maturation of mRNA:

Cleavage/poly A addition

1. What is the RNA cleavage/poly A signal?
• AAUAAA
2. With real human examples, discuss RNA cleavage/poly A errors.

Discuss the following mRNA modification that occurs during the maturation of mRNA:

RNA editing

1. Give a human example of RNA editing.
• In the human apolipoprotein-B gene there is a CAA codon at position 2154 that is targeted for editing.  Cytidine deaminase deaminates the C to U to form a UAA codon (stop codon).  Messages are edited in intestinal cells, but not in liver cells.

How can one gene produce more than one polypeptide?

Parts of polypeptides can be excised, with results in multiple polypeptides from one gene.  This is rare.