BIO STUDY GUIDE

Proofreading is backwards – 3’ to 5’! Enzyme goes and makes sure all bases match up. Removes nucleotides that aren’t accurately bound by polymerase. Exonuclease Alternative splicing and slicing are different

Strand is the coding strand that will be replicated 5’ GTAGCCTACCCATAGG 3’ coding strand broken3’ CATCGGATGGGTATCC 5’ template strand for RNA5’ GUAGCCUACCCUTUGG 3’ mRNA strand

How to read the chart: 1st codon – GUA = Valine2nd codon – GCC = Alanine3rd codon – UAC = Tyr4th Codon – CCA = Proline5th codon – UAG = Stop

Question 3: Look at translation picture with A E and P sites P site – entering­ Always have 3 tRNA molecules at a time

Will always starts at AUG sequence

Put in smooth strain and rough strain. Rough strain has no encapsulated outside so can be killed by mouses immunity. Killed one strain and put it iwith combination with live rough strains and found that mouse was still infected. Genetic material was being transferred from one strain to the next (which to which?) to infect the mouse.Have to be able to tell which strain is the leading strand and which is the lagging strandPrimer needed for replication!Ligase goes in and connects strands

Transcription takes place at ribosome ­ Open double helix

­ Take DNA and add RNA nucleotide bases to form mRNA

­ Know pre and post transcription and translation modifications

Letter’s experiment ­ Put DNA strand of a beta-globulin mixed with mRNA strand of beta-globulin. If hadn’t been spliced,

would have attached on top of each other. But what happened was – had mRNA strand down and found loops in the DNA strand, proving that for the DNA sequences in the loop, there was a spot missing the mRNA. Cuts off loop and reattaches to form mRNA.

Biofuel­ Electricity is much more effective and efficient

Microbiome­ Have co-evolved with other microorganisms symbiotically­ Microbiota diversity is influenced by topographical and temporal variation in the microbial communities­ Influenced by complex interactions through life such as diet, life-style, disease, and use of anitbiotics­ Relationship begins at birth – skin, mouth, vagina, intestinal­ 2/3 of our microbiota is unique to us­ Microbiota helps digest certain foods that the stomach and small intestine have not been able to digest­ Produces some vitamins (B and K)­ Immune system – forms a barrier effect­ Combats aggression from other microorganisms­ Specialized – Japanese can digest seaweed, we can’t

Prions­ Small infectious protein that is abnormally folded­ Causes progressive neurodegenerative conditions­ Act as a template and incite other proteins to fold abnormally as well­ Aggregated to form B-pleated sheets, forming amyloid plaque ­ Common in cannibal socieites, but also through bad meat, feces, blood, urine

­ Criteria for genetic materialo Contain the information necessary to construct an entire organisms (Blueprint for life)o Pass from parent to offspring and from cell to cell during cell divisiono Account for the known variation within and between species. That is, contain

information for niche-specific adaptation.­ Experiments determining DNA:

o Frederick Griffith’s work w/ Streptococcus and mice

Strains of Streptococcus pneumoniae secrete with capsules and have colonies that look smooth and can cause fatal infections in mice

Strains without capsule secretion have colonies that look rough and infections are not fatal

He injected heat killed-S bacteria and R-bacteria into mouse, causing the living type R cells to transform into virulent S cells

• Called transformationo Term used today for introducing DNA into cells

DNA from heat-shocked S-cells was transferred into the living type R bacteria, passing the capsule trait

­ Avery, MacLeod, and McCartyo Used purification methods to reveal that DNA is the genetic materialo Purified S-strain proteins, RNA and DNA and added each to R strainso Only purified DNA could transform type Ro Purified DNA might still contain traces of contamination that may be the transforming

principleo Added DNAse, RNAse, and proteases to ensure pure substanceso Mixed the DNA extract with R bacteria

Control +DNA +DNA, +DNAse +DNA, +RNAse +DNA, +Protease

o Added antibody to aggregate the R cells and remove them from suspension o R cells removed by centrifugation, plated the bacteriao Results showed no colonies on DNAse plate – proved that DNA is the genetic

material and is capable of transferring the information to make a capsule from the S strain to the R strains

­ Hersey and Chaseo Saw the T2 virus (bacteriophage) infects E. colio Phage coating was made completely out of protein that the DNA was found inside the

capsid (the top head of the phage)o Shearing force from a blender separated the phage coat from the bacteria

35P labels proteins 32P labels DNA

o The supernatant liquid either had 35S labeled empty phage or unlabeled empty phageo They measured radioactivity in supernatant liquid and found that DNA is the genetic

material in phages too

Describe and draw out the experiments that led to the identification of DNA as the genetic material. Who did these experiments and when? Be sure to include the controls for the experiments.

­ Nucleotideso 3 components- know structures!

Phosphate groups Pentose sugars (DNA vs RNA)

• Deoxyribose• Nucleoside – base linked to a sugar• Nucleotide – base linked to a sugar and one

or more phosphate groups Nitrogenous bases

• Purines- Adenine, guanine• Pyrimidines- Cytosine, Thymine/Uracil

­ Overall structure DNAo Nucleotides – building blocks of DNAo Single-stranded DNA (or RNA) is the active formo Two strands form a helixo DNA is packaged in an array of proteins (histones-to form chormosomes)o A genome is the complete complement of an organism’s genetic material

­ RNAo Ribose instead of Deoxyribose

DNA numbering

­ Sugar carbons 1-5o Base attached at 1’o Phosphate attached to 5’

­ Scientific discoveries for determining structureso Watson and Crick

Took Rosalind Franklin’s suggestion that DNA was a double helix Showed that it was an alpha helix using ball and stick models

o Erwin Chargoff showed that AT and GC were associated in pairs (Chargaff’s Rule) Draw a DNA molecule from the chromosomal to the molecular (nucleotide) level and everything in between. Include the structures of the nucleotides and the placement of the hydrogen bonding.

­ Structureo Nucleotides covalently bondedo Phosphodiester bond (phosphate group with two sugars to form the backbone, with

bases projecting)o NEGATIVELY charged because bases on the insideo Stabilized by hydrogen bondingo Double helixo Antiparallel strands

­ Space filling modelo Shows grooveso Major groove

Where proteins bind o Minor groove

­ Replicationo Meselson and Stahl experiment

Grew E coli in medium with only 15N then switched to only 14N, collected sample after each generations and observed line thickness

Proved semi-conservative theory!­ Bidirectional replication – replication that goes in both directions from the origin­ Eukaryotes vs prokaryotes replication

o Eukaryotes have multiple origins so DNA can replicate quicklyo Eukaryotic DNA is not circular like prokaryotes

­ Detailed process of replicationo Primase starts by forming primer, 5’ on the leading strand and 3’ on the lagging strand

Leading vs lagging strands• Lagging strand forms Okazaki fragments

o DNA helicase

Binds to DNA and travels 5’ to 3’ using ATP to separate strand and move fork forward

o Binding proteinso DNA polymerase adds nucleotides

In the form of deoxynucleoside triphosphates Unable to begin DNA synthesis on a bare template strand and can only work 5’ to

3’

o Ligase binds Okazaki fragments together after primers are replaced with nucleotideso DNA topoisomerase

Travels along helix and unwinds the DNA Diagram DNA replication from the lagging and leading strands. Be sure to add in all of the proteins involved.

­ Methods to prevent erroro Hydrogen bonding between A and T or G and C more stable than mismatcheso Active site of DNA polymerase unlikely to form bonds if pairs are mismatchedo DNA polymerase removes mismatched pairs (proofreading)

­ Telomeres!o The ends of chromosomes are very distincative short repeats f DNA

Special proteins bind to these repeats 3’ overhang at the end with no complementary strand Cannot copy this strand Telomerase saves the ends! Prevents chromosome shortening

• Attaches many copies of repeated DNA sequences to the ends of the chromosomes and

• Provides upstream site for RNA primer How do telomerases work?Diagram how DNA is replicated. What was the experiment done to show that replication is semiconservative? Who did this experiment

Genetic Expression­ Central Dogma of genetics

o Information cannot be transferred back from protein to either protein or nucleic acid. In other words, once information gets into protein, it can’t from back to nucleic acid

­ Garrod Hypothesiso Disease of black urine was due to missing enzyme, homogentisic acid oxidaseo Proposed relationship between genes and production of enzymeso Patients had recessive trait that caused high levels of homogentisic acid

­ Beadle and Tatum’s work with Neurospora Crassao Studied a common bread moldo Provided minimum requirements for growth and minimal mediumo Mutant strains would be unable to grow unless supplemented with some nutriento A single mutation resulted in the requirement for a single vitamino Next, isolated mutants that required arginine for growtho Three precursors of arginine

Orthinine, citruline, and then arginine Precursor molecule orthinine citruline arginine All areas of plate lacked a certain enzyme in the sequence except 4th plate

o Modifications since

Enzymes are only one ategory of cellular proteins and some are made up of proteins from several genes

More accurate – one gene encodes one polypeptide• Hemoglobin – 4 polypeptides required for one protein

Describe the experiments of Beadle and Tatum. How did they lead to the One Gene One enzyme theory? How has this theory been modified in more recent years?

Transcription­ Produces an RNA copy or transcript of a gene­ Structural genes produce messenger RNA (mRNA) that specifies the amino acid sequence of

a polypeptide o DNA RNAo Genes proteins traitso Promoter – site for RNA polymerase binding, signals beginning of transcription o Regulatory sequence – site for the binding of regulatory proteins. The role of regulatory

proteins is to influence the rate of transcription. Can be found in variety of locations o Terminator – signals the end of transcription

1. Initiation• Recognition step• In bacteria, sigma factor – causes RNA polymerase to recognize promoter region• In eukaryotes, a variety of transcription factors recognize promotes and direct RNA

polymerase• Catalytic portion of RNA polymerase has similar structure in all species

2. Elongation• RNA polymerase synthesizes RNA• Sigma factor is released at this time; increases specificity of RNA polymerase• Open complex (transcription bubble) is 10-15 base pairs long• Template (noncoding strand) is used for RNA• Syntehsized 5’ to 3’

3. Termination- RNA polymerase reaches termination sequence- Causes it an newly made RNA transcript to dissociate from DNA- Direction: synthesis vs reading

o Synthesis of RNA transcript is 5’ to 3’ and reads 3’ to 5’- Bacterial vs Eukaryotic

o Eukaryotic has more proteins per step

RNA polymerase II – transcribes the mRNA• Requires 5 general transcription factors to initiate transcription

RNA polymerase I and III – rRNA and tRNA - Eukaryotic mRNAs aren’t able to leave nucleus yet- Introns and exons

o Introns – parts of genes that do not code for proteins; internal sequences

Rare in prokaryoteso Exons – parts of genes that do code for proteins; become protein sequences

- Splicing – removal of intron by slicosome; exons reconnectedo Intron looped through snRNP

5’ splice site cut first 3’ splice site cut next

o Exons covalently reattached- Other modifications

o Addition of tails or caps

- Leder’s experiment proving splicingo Put DNA strand of a beta-globulin mixed with mRNA strand of beta-globulin

o If hadn’t been spliced, would have attached on top of each other

o Had mRNA strand down and found loops in the DNA strand, proving that for the DNA

sequences in the loop, there was a spot missing the mRNAo Cuts off loop and reattaches to form mRNA

- 5’ cap 3’ poly A tailo 5’ cap

7-methylgunosine covalently attached to 5’ end Important for

• Export from nucleus• Binding to ribosome

o 3’ poly A tail

100-200 adenine nucleotides added to 3’ end Increases stability and lifespan

What does it mean that the genetic code is degenerate? What is a reading frame?

- More than one codon can specify the same amino acid

- Preferred codon usage for certain species

- Stop codon doesn’t allow amino acid to bind

- Coding strand is the same sequence (not opposite)

Translation- Codons- 3 nucleotide bases

o 64 possible, third nucleotide is most variableo How to read a genetic code chart and convert it to a genomic sequenceo Start and stop codons- reading frames

AUG – Methionine, start• Defines reading frame

UGA, UAA, UAG – Stop- Role and structure of RNA helper molecules:

o mRNA

Has codon that code for amino acid sequenceo tRNA

Carries amino acids (tRNAser carries Serine) Clover leaf Anticodon – allows for binding of amino acid to mRNA Acceptor stem for amino acid binding

o rRNA

Ribosomal RNA Shapes ribosome

Describe the relative orientation of a gene on a chromosome, its mRNA and finally the corresponding protein.

Draw and label a tRNA and identify the important parts.

- How do amino acids attach to the proper tRNA?o Aminoacyl-tRNA synthetase catalyzes the attachment of amino acids to tRNA

o Two reactions result in aminoacyl-tRNA

Use ATPo Second genetic code

Ability of aminoacyl-tRNA synthetase to recognize appropriate tRNA

- Ribosomes- Eukaryotes vs. prokaryotes

o Prokaryotes have one kind of ribosome

30S + 50S 70So Eukaryotes have distinct kinds of ribosomes in different cellular compartments

40S + 60S 80S Describe the structure and function of ribosomes in Eukaryotes and Prokaryotes.

- A P E Siteso A site – amino acyl site; entry siteo P site – peptidyl site; addition siteo E site – exit site

- Subunits and assembled ribosomes (see above)- 3 Stages of Translation:

o Initiation

mRNA, first tRNA and ribosomal subunits assemble Requires help of initiation factors Requires GTP hydrolysis for energy input

o Elongation (3 Steps)

Synthesis from start codon to stop codon1. Aminoacyl tRNA brings a new amino acid to the A site

• Binding occurs due to codon/anticodon recognition• Elongation factors hydrolyze GTP to provide energy to bind tRNA to A site• Peptidyl tRNA is in the P site• Aminoacyl tRNA is in the A site

2. A peptide bond is formed between the amino acid at the A site and the growing polypeptide chain

• The polypeptide is removed from the tRNA in the P site and transferred to the amino acid at the A site – peptidyl transfer reaction

• Peptide bond formation is catalyzed by the rRNAo Ribosomal RNA is a ribozyme – RNA with enzymatic function

3. Movement or translocation or the ribocome toward the 3’ end of the mRNA by one codon

• Shifts tRNAs at the P and A sites to the E and P sites• The next codon is now at the A site• Unchanged tRNA exits from E site

o Termination

Complex disassembles at stop codon releasing completed polypeptide When a stop codon is found at the A site, translation ends 3 stop codons – UAA UAG UGA Recognized by release factors

- Eukaryotic vs. Bacterial differences in initiation

o Prokaryotes have mRNA that binds to small ribosomal subunit

The stop codon is a few nucleotides downstream Initiator tRNA recognizes start coon in mRNA Large ribosomal subunit associates AT the end of initiation the initiator tRNA is in the P site

o Eukaryotes instead of a ribosomal-binding sequence, mRNAs have 7-methylguanosine cap at 5’ end

Recognized by cap-binding proteinso Position of start codon more variable

In many cases, first AUG codon used as start codon- Release of sequence

o Completed polypeptide attached to a tRNA in the P site and stop codon in the A site

Release factor binds to the stop codon at the A site Bond between polypeptide and tRNA hydrolyzed to release polypeptide Ribosomal subunits and release factors dissociate

- Directionalityo N-terminus or amino terminus is first amino acido C-terminus or carboxyl terminus is last amino acid

Compare and contrast Eukaryotes and Prokaryotes as far as DNA replication, DNA structure and transcription and translation.

Gene Regulation- Why do we need to regulate genes?

o Making proteins takes up a lot of resourceso Unnecessary proteins may do cellular damageo “Constitutively-expressed genes”o Tissue-specific genes

- E coli and lactoseo When lactose is present, E coli makes lactose transporter (lactose permease) to

increase membrane permeability of lactoseo Proteins not made when lactose levels drop; increase when lactose in abundance

- Types of regulationso Transcriptional regulation

Small effector molecules• Can be metabolites• Bind to regulatory transcription factors and cause conformational changes

2 Domains in regulatory transcription factor can respond to small effector molecules

• Site where protein binds to DNA• Site for small effector molecule• Domains are functional regions of the protein• Effectors have some effect on cellular activities

Where does the bulk take place What is included in each type of regulation

- Operons

o Genes in bacteria are clustered in operonso Cluster of genes can be under transcriptional control of one promotero Transcribed into mRNA as polycistronic mRNA (coding sequences for 2 or more

structural genes)o Allows regulation of a group of genes with a common function

- Lac operon (positive and negative control)o E coli contains genes for lactose metabolismo Consists of promoter, LacI (regulatory gene), CAP site, lac promoter, operator, lacZ,

lacY, lacA and terminatoro 3 structural genes encoded in one long RNA

LacZ – B-galactosidase• Turns lactose into galactose and glucose through allolactose

lacY – lactose permease lacA – galactosidace transacetylase (unknown function)

o LacI

A negative regulator Regulatory gene – codes the lac repressor Not part of the operon because it has its own promoter

o Lac O – operator

Provides binding site for repressor protein o CAP Site

Activator protein binding site o Lac repressor protein binds to nucleotides of lac operator site preventing RNA

polymerase from transcribing lacZ, lacY, and lacAo RNA polymerase can bind but not move forward

- Allolactose o Small effector moleculeo Four molecules bind to lac repressor prevents repressor from binding to Operatoro Process called induction and lac operon is inducibleo Allolactose is made when a little galactose enters the cell and is acted on by B-

galactosidaseo As more lactose comes in, the removal of the repressors will increase

What are operons? What kinds of organisms have operons? What is the benefit of having genes in an operon?

- Jacob, Monod, and Pardee study with the lac repressoro Found rare mutants with abnormal lactose useo Expressed genes of lac operon constitutivelyo Some mutations were in the LacI region

Strains were called LacI- (normal strains are Lac L+) o 2 different functions for LacI region were proposedo LacI encodes a repressor protein

The LacI- mutation prevents the repressor from inhibiting the lac operon o Applied genetic approach using merozygotes

Merozygotes – partial diploids – a few genes have a second copy on a piece of DNA called and F’ factor

Strain of bacteria containinf F’ factor genes• Contain circular segments of DNA that carry additional copies of genes• In these mutants, some of them carry an extra copy of the lac operon and

a functional LacI geneo The mutant expressed the lac operon constitutivelyo The repressor shut down both copies of the gene in the absence of lactoseo Conclude that the LacI gene encodes a diffusible repressor protein

- Trans-effect – form of genetic regulation that can occur even though 2 DNA segments are not physically adjacent

o For example, action of lac repressor on lac operon- Cis-effect or cis-action element – DNA segment that must be adjacent to the gene(s) that it

regulateso For example, LacO site gives a cis effect

What experiment did Jacob and Monod perform to show the function of the lac repressor

Gene regulation can occur at several points between the gene and the functional protein. Describe these points and give an example of how regulation might occur at each.

- Repressible vs. inducibleo Lac operon is inducible because it is stimulated to turn on

CAP binds to CAP binding site and activates operono TRP is repressible because it is stimulated to turn off

When tryptophan levels are high, repressor binds to prevent transcription- Comparing bacteria gene regulation to eukaryotic regulation

o Prokaryotes

Microbes need to respond to changing food environment Most commonly occurs at transcription level

• Involved regulatory transcription factorso Bind to DNA in the vicinity of the promoter and affect transcription

of one or more nearby genes • Repressors inhibit transcription

o Exert negative control • Activators increase rate of transcription

o Exert positive control

Or control rate mRNA translated Or regulated at protein or post-translation

o Eukaryotes

Transcriptional regulation common too RNA processing Translation Post-translation Post-transcriptional regulation

o Post-translation for quick changes that need to be made- Eukaryotic transcription factors

o Eukaryotes need a more sophisticated method of regulation due to more specialized cells and proteins

o Activator and repressor proteins influence ability of RNA polymerase to initiate transcription

o Small effector molecules regulate tooo But

Genes almost always organized differently Regulation is more intricate Operons are rare in eukaryotes

o Activator proteins may promote the loosening of region in the chromosome where a gene is located making it easier for the gene to be recognized and transcribed by RNA polymerase

Describe the regulatory structure of the Eukaryotic promoter. Compare it to the promoters of prokaryotic genes.

- DNA methylationo Inhibits transcription, either by

Preventing the binding of an activator protein or By recruiting proteins that cause the DNA/chromatin to become more compact

o Shuts things downo Attaches methyl groups (DNA methylase) o Inhibits

- Eukaryotic structural geneso 3 features

Transcriptional start site • Where transcription begins

TATA box• 5’ TATAAAA 3’• 25 base pairs upstream from transcriptional start site• Determines precise transcriptional starting point (where RNA polymerase

binds) Response elements

• Recognized by regulatory proteins that control ignition of transcription - Eukaryotic transcription

o RNA polymerase II plus General transcription factors mountain plus mediatorso Preinitiation complex

Forms basal transcritption apparatus (GTFS and RNA polymerase II at the TATA box)

o Mediator

Partially wraps around GTFs and RNA polymerase II Mediates interactions with activators or repressor that bind to enhance silencers Controls rate at which RNA polymerase can begin transcription

• Activators stimulate faster initiation• Repressors inhibit progression to elongation

Regualtes• Activators (bind to enhancers)• Repressors (bind to silencers)

o Some activators binds to an enhancer and then influence function of GTFso May improve the ability of a GTF called TFIID to initiate transcriptiono Repressor may inhibit function of TFIID

- Epigeneticso Changes in gene expression caused by mechanisms not associated with DNA

sequenceo These can be inherited as cells divideo Most are not passed from generation to generation, but some can be if they are in the

sperm eggo Examples of epigentics

DNA methylation and chromatin remodeling microRNA Prion

o Can influence the phenotype without changing DNA - Accessibility – transcription is difficult if tightly packed- Some activators diminish DNA compaction near a gene and recruit proteins to loosen DNA

compaction- RNA silencing

o MicroRNAs (miRNA)

Hairpin loop structureo Short interfering RNAs (siRNA)o Small RNA molecules (22 nucleotides) that silence expression of mRNAso Partially completmentary to cellular mRNAs and inhibit translation by causing mRNAs to

be degradedo Bind to target mRNA with complementary sequence

Then degraded or translation is inhibited Gene silencing

- Alternative Splicingo When a pre-mRNA has multiple introns and exons, splicing may occur in more than one

way Alternative splicing causes mRNAs to contain different patterns of exons Allows same gene to make different proteins

o Acetyl-CoA opens proteins up

What is DNA methylation and how is it used to affect gene expression? What role does it have in DNA repair?

What are effectors and what is their importance in transcriptional regulation?

How many different kinds of hemoglobin are produced during the human life? What is the advantage of having so many kinds?

What are some transcription factor motifs? Where on the DNA molecule to they fit?

What is alternative splicing?