Genetics Exam 3

Be able to diagram and label a transcriptional unit in bacteria. (figure 13.6) What do the terms downstream and upstream mean? How do we number the nucleotides?

Upstream is toward the 5' end of the RNA molecule and downstream is toward the 3' end The first nucleotide transcribed is +1 (Downstream is positive, upstream is negative)

· How many nucleotides make up a codon? · How many possible codons does this make? · How many codons are stop codons? How many code for an amino acid? How many different tRNAs do most organisms actually have? · What is wobble and why is it necessary? · What does it mean when we say the genetic code is degenerate? · What is an isoaccepting tRNA? Why do we have them? Remember the genetic code is universal.

- 3 - 64 - 3 & 61 -30-50 When synonymous codons differ only in the third nucleotide. Flexible in bonding and still codes for the same amino acid -Amino acids can be coded by more than one codon (except tryptophan and methionine) -Isoaccepting tRNA: differing anticodons but the same amino acid. We have more codon probabilities than anticodon sequences

· Be able to draw the basic structure of an amino acid. · Remember that the R group is what makes the amino acids different. Also remember that they can be hydrophilic or hydrophobic

- Central Carbon - Hydrogen - Amino Group (NH3+) - Carboxy Group (COO-) - R group

Describe the structure of eukaryotic promoters. · What is the difference between the regulatory promoter and core promoter? What types of proteins bind to each? · How do transcriptional activators affect transcription? Do we get transcription in their absence? · What is an enhancer sequence? How is it positioned in relationship to the transcriptional unit? Remember when an activator protein binds and interacts with the BTA it causes the DNA to loop out. · What two common consensus sequences are found in the core promoter?

- Have different sequences to attract different polymerase types. Core: TATA box -25/-35 bp upstream, recognized by TFIIB. Regulatory: Located upstream of Core Promoter and has a mix and match of sequences -General Transcription Factors associate with RNA polymerase and Mediator to form BTA in Core Promoter. Transcriptional Activator Proteins bind to Regulatory Promoter -They increase the level of transcription, low levels are present in it's absence - Distant sequences that increase transcription -TATA and TFIIB recognition element

Explain elongation during transcription in bacteria. · What enzyme relieves strain of making DNA single stranded? Remember that as RNA polymerase moves along the DNA unwinding it with the transcript hanging out of the enzyme/transcription bubble complex. The DNA rewinds behind it.

-After 9-12 nucleotides, polymerase structure changes and is no longer bound to consensus sequences so it can move downstream. - Topoisomerase

Describe in detail what the end replication problem is and why it is a problem for linear chromosomes. · Describe how telomerase lengthens telomeres. · What two biomolecules make up telomerase? · What is the purpose of the RNA portion? · What types of cells have telomerase activity? What types of cells do not? If a cell lacks telomerase activity, what does this mean in terms of the number of times the cell can divide?

-After the primer is removed from the 5' end of the chromosome, there is no free -OH group -Chromosomes would shorten with each replication, removing telomeres and destabilizing chromosome 2. Telomerase is a ribonucleoprotein that adds an extra sequence and extends the 3' end, preventing telomere shortening 3. RNA and protein 4. RNA portion provides complementary nucleotides 5. Stem cells, germ cells, single-celled organisms -Mature cells dont have telomerase activity (somatic cells have a limited number of replications)

Describe Beadle and Tatum's experiments with Neurospora crassa that allowed them to work out different metabolic pathways. (We concentrated on the pathway that generates arginine.) · How did they induce mutations? · Remember auxotrophic vs. prototrophic. · Is the model organism they used haploid or diploid? What are the implications of this? Remember that these studies lead to the hypothesis that one gene/one polypeptide.

-Determined specific effect of mutation and worked out metabolic pathways. - Induced mutations by irradiating spores - Prototrophic: wildtype can grow on minimal media -Auxotrophic: mutant can grow on complete media (that contains its missing nutrient) - Haploid. They can directly see mutations in the following generations.

You should also know everything about how operons are regulated including but not limited to: · Describe the difference between: inducible vs. repressible, negative vs. positive regulation. · Now you should be able to put these together and describe how each of the four types of operons are regulated: is the regulator protein synthesized in its active or inactive form? What makes it active or inactive? What two domains does the regulator protein have? · Why is it advantageous for the bacterial cell to organize structural genes into operons?

-Negative Control: Regulatory protein has a repressor function -Positive Control: Regulatory protein has an activator function -Inducible: Transcription is usually off -Repressible: Transcription is usually on Is the regulator protein able to bind to the operator immediately? -RNA binding domain and effector domain -Product is only synthesized if needed, saving energy

Make sure you can explain and diagram (including labeling) everything in the ENTIRE process of replication in bacteria. Figure 12.10c, 12.12, 12.13, and 12.14 helpful. · Understand the directionality of synthesis. Why is one strand synthesized continuously (the leading strand) and one discontinuously (lagging) at a replication fork? (remember these are the new strands not the template) What are the fragments called? Be able to identify each type of strand. · Also remember that the strand being synthesized is complementary and antiparallel to the template. And that both strands are synthesized at the same time. · Where does the process start? How does it start? · Function of: Helicase, ss binding proteins, DNA gyrase, primase, DNA polymerase III and I (explain function of ALL enzymatic activities for these), & ligase. (Why is ligase necessary?) · Why are primers necessary? What type of polymerase is primase (what nucleotides make up primers)? · Remember the rest of DNA polymerases in E. coli pretty much function in repair. What do all of the DNA polymerases have in common?

-New DNA is made by enzymes called DNA polymerases, which require a template and a primer (starter) and synthesize DNA in the 5' to 3' direction. During DNA replication, one new strand (the leading strand) is made as a continuous piece INTO the replication fork. The other (the lagging strand) is made in small pieces OUT of the replication fork (Okazaki fragments). DNA replication requires other enzymes in addition to DNA polymerase, including DNA primase, DNA helicase, DNA ligase, and topoisomerase. -The strand being synthesized is complementary and antiparallel to the template. -Both strands are synthesized at the same time Helicase: Breaks hydrogen bonds between the two strands and unwinds DNA at the replication fork ss Binding Proteins: Prevent secondary structure formation and stabilize single strands DNA gyrase: (topoisomerase) releases tension ahead of replication fork DNA Polymerase III: Main enzyme that carries out replication, Adds nucleotides to an existing 3' end. Has 3'-5' exonuclease proofreading activity. High processivity. DNA Polymerase I: Removes and Replaces primers with 5'-3' exonuclease activity DNA Ligase: Seals the nicks between discontinuous strands using a phosphodiester bond -DNA polymerase cant initiate a new strand - it can only add nucleotides to an existing 3'-OH end (Primase is an RNA Polymerase made of RNA nucleotides) DNA Polymerases Have in Common: -Use the template to determine the order of nucleotides -Synthesize in the 5'-3' direction -Use dNTPs - Require a primer -Catalyze phosphodiester bond by joining 5' phosphate of new nucleotide to the 3'-OH group of the preceding nucleotide

Describe the 3 challenges that eukaryotic replication has in comparison to bacteria. Explain how each of these is overcome. · How is the challenge of genome size overcome? · Describe, in detail, how a eukaryotic cell ensures that the genetic material is replicated once, and only once during S phase. · What is the licensing factor?

1. DNA is associated with histones: nucleosome assembly immediately follows DNA replication 2. Eukaryotic genome is much larger - ensure replicated once and only once during S phase 3. Linear chromosomes: shortening of telomeres -During DNA replication, nucleosomes ahead of the replication fork must be disassembled to facilitate the movement of the DNA replication machinery, and behind the fork, new nucleosomes must be reformed on daughter strands -Multiple Origins of replication make it more efficient and faster -ORC binds to the origin and provides the foundation for the assembly of pre-replication complex -cdc6, which is only produced during G1 phase, binds to ORC -cdc6 must bind before MCM can bind: helicase that unwinds DNA to initiate replication -MCM must bind for replication to occur. Once it is bound to the origin it is said to be "Licensed for replication" -Once replication is initiated, MCM and cdc6 are displaced * cdc6 is rapidly degraded; preventing further initiation by MCM binding * Geminin also prevents MCM from binding to DNA * Geminin is degraded at the end of mitosis to allow MCM to relicense origins

Post-transcriptional modifications for eukaryotic pre-mRNA: · 5´ cap: details of structure (figure 14.6), when is it added, 2 functions? · Poly(A) tail: structure, when is it added, & 2 functions? · Remember there is a consensus sequence that determines the cleavage site. · Splicing: Where does it occur? Function? What sequences are required? Describe how an intron is spliced out including the 5 snRNPs. What unique bond is formed when a lariat structure is generated? What happens to the lariat

1. Guanine + phosphate is added to remaining two phosphates on 5' end. Nucleotides at that end are methylated. Added first. Stabilizes molecule and allows for initiation of translation 2. Cleaved on the 3' end between two consensus sequences and then adenine nucleotides are added (polyadenylation). Added 2nd. Stabilizes molecule and aids in ribosome attachment. 3. Splicing of introns occurs at three points; the 5' (GU) & 3' (AG) splice sites and the branch point (an adenine towards the 3' end). Takes place within the spliceosome with snRNPs. Happens last. 5' intron is cut (U1) and folds back on itself at the branch point. 3' intron is cut (U2, U5, and U6 make up spliceosome) and intron is released. 3' of exon 1 ligates with 5' of exon 2 Phosphodiester Bond It reverts to linear form and gets degraded

· Be able to describe/diagram the entire process from transcription to mature mRNA formation (slide 18)

1. Introns, exons and a long 3' end are all transcribed into pre-mRNA 2. A 5' cap is added 3. Cleavage occurs at the 3' end approx. 10 nucleotides downstream of consensus sequence 4. A polyA tail is added 5. Introns are removed 6. Mature mRNA

Explain initiation of transcription in bacteria. Don't forget to include: · Steps · Be able to explain promoter structure & function (what information is embedded in this region)? Why is the promoter so important? · What happens if there's a mutation in a consensus sequence (elements)? · What happens if the elements are moved upstream or downstream? · What are two important promoter elements in bacteria? Where does unwinding of DNA occur?

1. Promoter recognition by transcriptional apparatus. Formation of transcription bubble. Creation of first bonds between rNTPs. Escape of transcriptional apparatus from promoter. 2. Sequences of DNA recognized by the transcriptional apparatus with essential information about where transcription starts and which strand is read in what direction. Contain consensus sequences. 3. Can alter the rate of transcription 4. RNA polymerase won't be positioned correctly 5. -10 and -35 consensus sequences 6. -10 sequence

Explain the function of the six classes of RNA that we focused on.

1. Ribosomal RNA (rRNA) -Joins with protein subunits to form ribosomes (site of polypeptide synthesis) 2. Messenger RNA (mRNA) -Primary structure codes for the amino acid sequence of a polypeptide *Pre-messenger/primary transcript is the immediate product of transcription in eukaryotes (Needs to be extensively modified before exiting the nucleus) *Prokaryotic mRNA begins to be translated before transcription is even complete 3. Transfer RNA (tRNA) -Brings specific amino acid to the ribosome for incorporation into the growing polypeptide 4. Small Nuclear RNA (snRNA) -Joins with small nuclear proteins to form snRNPs- small ribonucleoproteins *Assist with post-transcriptional modifications of primary transcript in eukaryotic cells (splices out introns) 5. Small Nucleolar RNA (snoRNA) -Aids in the processing of rRNA in eukaryotic cells 6. Micro RNA (miRNA) and Small Interfering RNA (siRNA) -Activates RNAi (RNA Interference) -Initiates degradation mRNA molecules or blocks translation (regulates gene expression)

You should be able to explain and/or diagram the overall process of transcription in bacteria. (Figure 13.4 & 13.8) · Directionality of transcription? Direction the DNA is read? Remember the RNA is complementary and antiparallel to the DNA template as it is being synthesized. · What are the requirements for transcription? · Compare and contrast replication and transcription. Difference between the template and nontemplate strand? How is the template strand determined? Remember that the RNA that results from transcription has the same sequence as the nontemplate strand except it has U's instead of T's

1. Sigma factor associates with the core enzyme to form a holoenzyme which binds to the -35 and -1 consensus sequences in the promoter. The holoenzyme binds to the promoter tightly and unwinds the dsDNA. A nucleoside triphosphate serves as the first nucleotide in the RNA molecule. Two phosphate groups are cleaved and sigma factor is released as the RNA polymerase moves beyond the promoter. 2. 5'-3'. DNA is read 3'-5' 3. ssDNA template, ribonucleoside triphosphates, transcription apparatus

List and describe the four stages of translation in bacteria. (Figure 15.21) Be sure and include: · tRNA charging: enzyme? How is it specific? What is formed as a result? Remember it is the carboxyl group that attaches to the tRNA. This will be important with the directionality of protein synthesis. ** · Initiation: How is the mRNA recognized by the small subunit in E. coli? Why is IF-3 important? What is the first aminoacyl-tRNA? When does the large complex come in and bind? · Describe the structure of the ribosome after initiation. What are the three sites called? · Elongation: Describe the three steps. · Which site holds the incoming charged tRNA? · What enzyme activity transfers the growing polypeptide? Explain how this occurs and relate to the directionality of protein synthesis. ** · Describe and diagram the process of translocation including the three tRNA sites for the ribosome and what goes on in site. How do these shift as translocation occurs? (Be able to describe in detail figure 15.19.) · Termination: When does termination occur? Remember releasing factors participate in termination. · Also remember that GTP is utilized as an energy source for the process of translation.

1. tRNA Charging: aminoacyl-tRNA synthetases. Recognizes different sequences on tRNA and binds appropriate amino acid. Forms aminoacyl-tRNA. 2. Initiation: IF-3 binds small subunit and then binds to Shine-Delgarno on mRNA. IF-3 keeps small subunit from binding to large. fMet-tRNAfmet. Large subunit binds after IF dissociates. 3. Elongation: A-P-E -A -Peptidyl transferase 4. Termination: When stop codon enters A site

What comprises the core RNA polymerase in bacteria? What about the holoenzyme? Does it require a primer? · Why is sigma factor necessary? Is there just one? · Remember there is a single RNA core polymerase for bacteria

5 polypeptide subunits 5 subunits + sigma factor No It helps recognize and bind to the correct place on the promoter. There are multiple.

Be able to diagram mRNA structure and explain the functions of the parts. (figure 14.5) · What's the Shine-Delgarno sequence? (remember this is only found in bacteria)

5' untranslated region: Doesn't code for AA (Shine Delgarno sequence: ribosome attachment) Start Codon Protein-coding region: codes for AA Stop codon 3' untranslated region: Stability

Know the functions of eukaryotic DNA polymerase alpha, delta and epsilon

Alpha: Primase Activity (DNA pol I) Delta: Lagging Strand Synthesis (DNA pol III) Epsilon: Leading Strand Synthesis (DNA pol III)

· What two ways can pre-mRNAs be alternatively processed? What implications does this have on protein structure after translation

Alternative Splicing and Multiple 3' Cleavage Sites It is not what was transcribed so RNA editing occurs. Different mature mRNAs each result in a different polypeptide.

Be able to describe initiation of transcription in eukaryotic cells (figure 13.17 in 4th edition 13.16 in 5th, 6th, and 7th edition) Include all the elements and proteins shown

Assembly of transcriptional factors. 1. TFIID binds to TATA box in the core promoter (TBP is the binding protein) 2. Transcription factors and RNA polymerase II bind to the core promoter where RNA pol II is positioned over start site 3. TAP bind to sequences in enhancers and the DNA loops out to allow the enhancer to interact with the BTA 4. Transcriptional Activator Proteins bind to sequences in the regulatory promoter and interact with BTA through mediator

· Why are proteins important? What functions do they serve?

Central to all living processes -Structural, Regulatory, Communication, Defense litrelly erything babes

Be able to write out the precise definition of a gene

DNA sequences that are transcribed into a functional RNA molecule

· Be able to explain why controlling gene expression is VITAL to multicellular organisms and single cellular organisms in detail.

Development and differentiation in multicellular organisms: specialized cells need proper gene regulation Response to Environment: Maintaining Homeostasis - prokaryotes respond to availability of nutrients, eukaryotes respond to changes in the internal environment Bacteria will only produce certain proteins at any given time

Explain how transcription is different in eukaryotes compared to prokaryotes. (monocistronic vs. polycistronic)

Eukaryotes transcribe one gene at a time per one promoter 3 different RNA polymerases

· How does eukaryotic initiation differ from prokaryotic initiation?

Initiation Complex: 5' Cap + Initiation Factors (7) + small ribosome subunit cap-binding proteins, helicase activity and polyA tail No Shine-Delgarno Sequence

What are introns and exons? Remember bacteria typically do not have introns. Know how many introns are in a pre-mRNA if given the number of exons.

Introns: Intervening sequences that are spliced out of pre-mRNA before leaving the nucleus (Group I & II: Self-Splicing) Exons: Coding sequences Ex. If 4 exons then 3 introns

· What are the posttranslational modifications that can take place after the polypeptide is synthesized?

Methionine can be removed polypeptide chain can be cleaved Carbohydrates can attach AA can be modified Folding into 3D shape (chaperone proteins)

Be able to diagram and label the parts of the lac operon and describe how it is regulated. Why is this kind of regulation advantageous to the cell? · What is lactose? · Be sure and include the structural and regulator genes and their known functions for lactose metabolism.

Negative Inducible Promoter-Operator-lacZ-lacY-lacA lacZ: Beta-galactosidase, breaks lactose into glucose and galactose lacY: Channel Protein In the absence of lactose, the regulator protein binds to the operator and inhibits transcription

Be able to describe and diagram operon structure and the regulator gene. Is the regulator gene a part of the operon? Where is it located? · Describe the difference between polycistronic and monocistronic · How are bacterial and eukaryotic genomes organized with respect to these terms?

Operon: Promoter, Operator, and Genes P: site for RNA pol binding O: on/off switch -Regulator Gene is not a part of operon; has it's own promoter -Polycistronic: One promoter for a group of structural genes (Bacteria) Eukaryotes are Monocistronic

· Explain briefly the four levels of protein structure. · What interactions does tertiary structure depend on? Which one is the most important? Leading to: What happens if a mutation causes a hydrophobic amino acid to be substituted for a hydrophilic one in a protein? Or visa versa? See sickle cell anemia example.

Primary: Amino Acid sequence Secondary: Beta-Pleated Sheets or Alpha Helices Tertiary: 3D conformation; secondary structures interact and fold (determined by amino acid sequence (R group interactions) - hydrophilic/hydrophobic) Quarternary: Multiple polypeptide subunits associate to make a complete protein If the critical amino acid is changed the entire protein structure can be effected which is necessary for the function of the protein

Explain Rho independent and Rho dependent termination in bacteria. · What steps must occur for termination to take place? · Why is termination necessary? · What enzyme activity does Rho have?

RHO Independent: Inverted complementary sequences that form a hairpin loop when transcribed that slows transcription. Repeat polyA causes RNA to pause. Weak, so transcript separates from DNA template. RHO Dependent: Rho factor binds to transcript region with no secondary structures and moves towards 3' end. When terminator is transcribed RNA polymerase pauses and rho factor catches up to separate RNA from DNA - RNA polymerase must stop, dissociate from DNA, and detach - Helicase activity

Which RNA polymerase transcribes pre-mRNAs in eukaryotic cells?

RNA polymerase II

· Define regulatory gene, structural genes, regulatory elements and constitutive expression. · Remember that proteins need to have a DNA binding domain to bind to DNA.

Regulatory Gene: Code for RNA/proteins that affect transcription/translation (DNA binding proteins) Structural Gene: Code for proteins involved with general processes (enzymes or structural components: glycoproteins, receptors) Regulatory Elements: Sequences of DNA that are not transcribed; site of binding to regulatory proteins Constitutive Expression: Not regulated; expressed all of the time (Housekeeping)

Define replicon. How many do bacterial chromosomes have? Why do eukaryotic genomes have 1000s

Replicon: Individual unit of replication Bacteria have a single circular chromosome Eukaryotes have multiple chromosomes

What is the reading frame of a mRNA? What direction is the mRNA read? Remember protein synthesis occurs from the N term end to C terminal end.

Start codon to Stop codon. 5'-3' N-C

· Describe the processes of RNAi. · Remember double stranded RNA triggers the process. · What is the function of slicer? What is the RNA induced silencing complex? · Compare and contrast miRNA with siRNA.

Suppression of gene expression. - Dicer cleaves dsRNA. One strand associates with RISC (RNA-induced silencing complex). RISC pairs with mRNA with a complementary sequence. -Slicer: endonuclease - miRNA: found in all eukaryotes. Inhibition of translation. Forms short hairpins of dsRNA. Targets non-complementary genes. - siRNA: originates from transposons or viruses. RNA degradation. Long dsRNA hairpins. Targets complementary sequences.

What is RNA editing? What RNA molecule participates

The altered mRNA sequence after transcription is edited by adding, deleting, or changing bases with the help of guide RNA (gRNA)

Describe the three types of replication and know what type of DNA utilizes each. Compare and contrast these

Theta Replication: (Circular DNA) -Replication bubble and fork; bidirectional Rolling Circle: ( Special Plasmids) -Unidirectional Linear: (Eukaryotes) - Bidirectional; multiple origins of replication

What is special about group I and group II introns

They are self splicing

How does chromatin structure impact transcription? What enzymes modify chromatin structure?

Transcription requires DNA to be accessible so nucleosomes must be altered. HATs acetylize histones to reduce the positive charge

Describe Meselson and Stahl's experiments that determined DNA replicates in a semi-conservative manner. · Explain their results and how the other two methods were ruled out. · What does semi-conservative mean?

Two Nitrogen isotypes: 15N (heavy) and 14N (common) Used equilibrium density gradient centrifugation After one round of replication; the intermediate band After the second round of replication; two shortly spaced bands After the third round; the Same short spaced bands but lighter is darker DNA separates into two strands, and each strand serves as a template; each new molecule consists of one old strand and one new strand

· What is a polyribosome? Remember that eukaryotic cells and prokaryotic cells can have multiple ribosomes translating the same mRNA at the same time. However, transcription and translation cannot take place at the same time in eukaryotes. Why?

a group of ribosomes moving one after another and translating the same mRNA chain is formed. -Posttranscriptional modifications must occur before mRNA can leave nucleus to be translated

· Describe the structure of tRNA. Be able to sketch out a basic 2D structure and label the acceptor arm (Where the amino acid attaches) and anticodon arm. · Remember the acceptor arm has the same three nucleotide sequence at the end for all tRNAs.

"Clover Leaf" 3' Acceptor arm on top right (CCA) Anticodon region at the bottom point

What makes up the eukaryotic BTA? · What is the function of the mediator? Remember that the general TFs facilitate RNApol II binding to the promoter and initiation of transcription. Know the function of TBP (a part of TFIID) and TFIIB

1. RNA Polymerase II + General Transcription Factors + Mediator 2. Communicate between BTA and activator proteins 3. TBP: A part of TFIID that binds TATA TFIIB: Promoter Recognition

What is the difference between prokaryotic and eukaryotic mRNA? What does that mean with respect to the timing of translation

Prokaryotes don't typically have introns so they can start the process of translation before transcription is even complete. Eukaryotes have to extensively modify the pre-mRNA/Primary Transcript before it can leave the nucleus to be translated; which can include splicing introns, 5' capping, and PolyA tail

How is transcription terminated in yeast cells? How does this differ from bacteria

RNA pol II transcribes well past the coding sequences of most genes and a cleave is made near the 3' end of the RNA (at a specific site consensus sequence) while RNA pol continues transcribing, Rat1 exonuclease attaches to the 5' end of the trailing RNA and moves towards the RNA pol while degrading the RNA, once Rat1 reaches RNA pol II termination is complete. Cleavage site is encoded, no secondary structures, continuation of transcription instead of pausing.

What are the basic requirements for replication

Single-stranded DNA template deoxyribonucleoside triphosphates Enzymes and other proteins (DNA polymerase)

Why are dNTPs needed for synthesis? (Why the three phosphates instead of just one?)

dNTP provides nucleotides to the "unzipped" strand using the template of a single side. These phosphates play key roles in the addition of subsequent nucleotides to the daughter strand.