Cell Structure Final Exam New Material

* What is the genetic code?

The relationship between DNA base sequences and the linear order of amino acids in proteins

How many DNA bases are there and how many amino acids are there?

4 DNA bases, 20 amino acids

1. * Why is DNA not a doublet code?

2. How many combinations are possible in a triplet code?

3. * What is the function of the triplet code?

1. 2 bases specifying a single amino acid would be inadequate because only 16 combos would be possible.

2. 3 bases per amino acid = 64 possible combos, which is more than enough for all 20 amino acids

3. Translating an mRNA sequence into the correct amino acid sequence

1. * What is a codon?

2. How many codons specify specific amino acids?

3. What codon is the START codon?

4. Which codons are the STOP codons?

1. A sequence of 3 nucleic acids coding for 1 amino acid (a triplet code)

2. 61/64 specify specific amino acids (all of them + START)

3. AUG (Met amino acid)

4. UAA, UAG, UGA (not amino acids)

Is the genetic code universal for all organisms?

Yes, except for a few rare exceptions

* What does it mean when we say the genetic code is unambiguous and degenerate?

Unambiguous: every codon has one meaning only

Degenerate: Many of the amino acids are specified by more than one codon

* What is the directional flow of genetic information?

The Central Dogma of Molecular Biology

• DNA is the template for RNA synthesis, which directs protein synthesis

1. Which enzyme catalyzes reverse transcription?

2. What are viruses that perform reverse transcription called?

1. Reverse transcriptase: an RNA-dependent DNA polymerase

2. Retroviruses (ex: HIV)

* Generally describe transcription and translation.

• Transcription: RNA synthesis using DNA as a template

• Translation: synthesis of proteins using information in RNA

* What are the three major products of RNA transcription, and what are their functions?

• mRNA: RNA that’s translated into protein

• rRNA: integral component of ribosome

• tRNA: brings amino acids to ribosome

* Q: Mutations that alter sequences near the 5’ end of mRNA result in alterations near the corresponding protein’s N-terminal end, whereas mutations that alter the 3’ sequences of mRNA result in alterations in the protein’s C-terminal end. What do these findings imply?

The order of nucleotides from 5’ to 3’ in mRNA determines the order of amino acids from the N- to C- termini.

What are the characteristics of RNA?

• Ribose instead of deoxyribose

• Uracil instead of thymine

• Single-stranded, flexible

• Can form various structures by complementary base pairing within single strands

1. * Which strand is read during mRNA synthesis? What is the other strand called?

2. * What direction does transcription progress?

1. The template strand is read to create complementary RNA strand; the other strand is the coding strand (same sequence as the mRNA with T instead of U).

2. Progresses in the 5’ to 3’ direction, like DNA, with each nucleotide being added to the 3’ end.

1. * True or False: only a subset of the DNA in any organism codes for protein and is therefore transcribed and translated. What are the other portions of DNA called?

2. * Why is this important?

1. True; Noncoding regions

2. This is why the process needs to be regulated (so the entire genome is not transcribed inefficiently)

* Describe the basic elements required for transcription (in the transcription unit: DNA components that give rise to 1 RNA molecule):

1. DNA-dependent RNA polymerase

2. Promoter

3. Transcription start site (TSS)

4. Start codon

5. Stop codon

6. Terminator sequences

1. DNA-dependent RNA polymerase: An enzyme which uses DNA as a template to add new ribonucleic acids on a growing strand of RNA

2. Promoter: A seq of DNA that can decide where RNA polymerase binds

3. Transcription start site (TSS): A seq of DNA that can decide where transcription starts

4. Start codon: A coding seq to start the 1st amino acid (usually AUG)

5. Stop codon: A coding seq to stop the addition of amino acids (tells ribosome to stop translation) - does NOT stop transcription

6. Terminator sequences: A seq of DNA to stop transcription

* What are the four stages of transcription, and what are their general processes?

1. Binding: RNA polymerase binds to a promoter seq, triggering local unwinding of the double helix (transcription bubble)

2. Initiation: RNA polymerase initiates the synthesis of RNA using 1 DNA strand as a template

3. Elongation: RNA polymerase moves along the DNA template, unwinding the helix & elongating the RNA

4. Termination: RNA polymerase dissociates from the DNA template, leading to termination of synthesis & release of the RNA molecule

1. * A typical bacterial promotor has specific _________ elements.

2. * What is the function of these?

1. Upstream (UP)

2. They have certain DNA base sequences which facilitate the binding of RNA polymerase subunits

* How many types of RNA polymerase do bacteria have?

Just one - it synthesizes all major RNA classes.

1. What is the difference between the bacterial RNA polymerase core enzyme and holoenzyme?

2. * What does the bacterial RNA polymerase sigma (σ) factor/subunit do?

1. Core: lacks sigma subunit & can carry out RNA synthesis; Holo: includes all subunits & is required to initiate transcription

2. Helps RNA polymerase bind to DNA to initiate transcription, then dissociates as the transcription bubble shrinks to allow elongation to proceed

* Describe what happens as RNA polymerase elongates the RNA and moves along the DNA strand.

As RNA polymerase moves along the DNA strand during elongation, the double helix is ahead of the polymerase is unwound, and the DNA behind it is rewound back into a double helix

Can RNA polymerase proofread?

Yes, it can back up slightly to replace an incorrect nucleotide with a correct one: RNA backtracking

1. * When will RNA elongation during bacterial transcription terminate?

2. * What happens then?

1. Until RNA polymerase copies a sequence called the termination signal.

2. As the termination signal is transcribed into RNA, it folds into a 3D shape (hairpin loop) which helps the polymerase dissociate from the DNA & releases the RNA

* Q: A DNA sequence might be the promoter that drives the expression of a dynein motor gene in bacteria. You make a mutation that removes the sequence TATATAT from the -25 region of this putative promoter. If the original sequence serves as a promoter, what should happen to the transcription of the dynein motor gene in the mutant?

Transcription should decrease (RNA polymerase would have a difficult time recognizing the promoter)

* What are the ways in which eukaryotic transcription is more complex than bacterial transcription?

1. There are additional transcription factors needed.

2. There are 3 RNA polymerase enzymes instead of one, which transcribe one or more different classes of RNA.

3. There are more varied RNA promoters (one for each type of RNA poly).

4. RNA is cleaved and processed after transcription. (Bacterial DNA doesn’t have introns)

* What are the three types of RNA polymerase in eukaryotes, where are they located, and what are their functions?

1. RNA Polymerase I: In the nucleolus, synthesizes an RNA molecule that is a precursor for 4 types of rRNA

2. RNA Polymerase II: In the nucleoplasm, synthesizes mRNA

3. RNA Polymerase III: In the nucleoplasm, synthesizes tRNA and the small rRNA

* During eukaryotic RNA cleavage, what are the roles of introns and exons?

* Pre-mRNA contains coding (Exons), which are kept, and noncoding regions (Introns), which are spliced out to make the mature mRNA.

1. * What do eukaryotic general transcription factors do?

2. * What complex do they eventually form with RNA Pol II?

1. Bind DNA elements first, then recruit RNA polymerase to promoters on DNA to initiate transcription.

2. Many transcription factors bind the promoter in a defined order to eventually form a large protein complex with RNA Pol II, called the pre-initiation complex

* During transcription initiation in eukaryotes, what is the TATA box?

DNA sequence found in the core promotor, which certain transcription factors (containing a TATA-binding domain) specifically bind.

* During transcription initiation in eukaryotes, what does the general transcription factor TFIIH do?

It’s a helicase and a kinase:

• Locally unwinds the DNA

• Phosphorylates the C-terminal tail of RNA Pol II

Both activities release the RNA Pol II from the initiation complex to start transcription elongation.

What do additional short sequences upstream of the promoters in eukaryotes do?

Proximal control elements and enhancer elements/distal control elements can improve the promoter’s efficiency.

* What nucleotide sequence mediates the termination of RNA synthesis in eukaryote transcription, and what does it do?

The AAUAAA sequence causes the cleavage (termination) and recruits the polyadenylation complex to the mRNA.

* Q: The relationship between general transcription factors and the TATA-binding protein is…?

The TATA-binding protein is a type of general transcription factor.

1. What is eukaryotic mRNA processing of the primary transcript for?

2. * When does RNA processing begin?

1. To stabilize mRNA (increase time until degradation)

2. Cotranscriptionally (the C-terminal domain of RNA polymerase can bind enzymes for capping, splicing, cleavage, and polyadenylation)

* What is added to pre-mRNA during processing and at which ends?

1. 5’ Capping: capped with a modified Guanosine at the 5' end (the AUG end)

2. 3’ Polyadenylation: addition of a series of A’s added to the 3' end - Poly(A) tail

* What RNA-protein complexes are many mRNAs spliced by?

Spliceosomes

* What does the presence of introns allow for during splicing?

Allows for alternative splicing: each gene’s pre-mRNA can be spliced in multiple ways to make multiple unique mature mRNAs

* Q: Which of the following types of RNA in eukaryotic cells come(s) from precursors subject to RNA processing: rRNA, tRNA, mRNA, or all?

All of the above (rRNA, tRNA, and mRNA)

* Q: Where in the cell is rRNA synthesized?

In the nucleolus inside the nucleus.

* Why can bacterial translation occur co-transcriptionally (at the same time as transcription)?

1. Because there is less RNA processing required

2. Bacteria don’t have a nuclear envelope, so there is no physical separation of the two events, like there is in eukaryotes, where transcription occurs in the nucleus, translation in the cytoplasm

* Describe the roles of the following in the process of translation:

• Ribosomes

• tRNAs

• Aminoacyl-tRNA synthetases; how many are there?

• mRNA molecules

• Protein factors

  • Initiation factors

  • Elongation factors

1. Ribosomes: carry out polypeptide synthesis

2. tRNA: align amino acids in the correct order based on the mRNA & bring amino acids to the ribosome

3. Aminoacyl-tRNA synthetases: enzymes that attach amino acids to the appropriate tRNA molecules (charge the tRNAs) - 20 of them, 1 for each amino acid

4. mRNA molecules: encode amino acid sequence information

5. Protein factors: facilitate some steps of translation

   1. Initiation Factors (IFs): initiate protein synthesis

   2. Elongation Factors (EFs): elongate protein synthesis

* What is the overall function of ribosomes and what are they made out of?

Function: to carry out polypeptide synthesis

Composed of: rRNA & protein, large & small subunits

* Describe the particular steps of translation which occur at each of the following parts of the ribosome:

1. mRNA-binding site

2. A (aminoacyl) site

3. P (peptidyl) site

4. E (exit) site

1. mRNA binding site: where the ribosome binds & positions the mRNA so that codons can be read in the correct 5' → 3' direction. It aligns the start codon in the P site to initiate translation.

2. A (aminoacyl) site: binds a new aminoacyl-tRNA (tRNA with its amino acid) whose anticodon matches the next codon on the mRNA.

3. P (peptidyl) site: holds the tRNA carrying the growing polypeptide chain. Peptide bond forms between the chain in the P site and the new amino acid in the A site. Then the new tRNA with the amino acid chain moves over to the P site.

4. E (exit) site: After the peptide chain is transferred, the now-empty tRNA moves to the E site. Where uncharged tRNA exits the ribosome.

* What are the two most important sites on a tRNA molecule?

1. The amino acid attachment site at the 3’ end that attaches to a specific amino acid

2. The anticodon loop which is complementary to a codon in the mRNA molecule.

* Which base usually occupies the wobble (or third) position in the tRNA anticodon loop and why?

Inosine, because it’s able to pair with U, C, or A and allows for several codons to specify one amino acid (degeneracy)

1. What is a tRNA linked to an amino acid called?

2. How do we describe that tRNA?

3. How do we describe that amino acid?

1. Aminoacyl tRNA

2. Charged

3. Activated

* Translation begins at the __-terminus of the polypeptide & adds amino acids to the growing chain until the __-terminus is reached.

N, C

* What are the 3 stages of translation?

1. Initiation: components of the translational apparatus come together with an mRNA; tRNA carrying the 1st amino acid binds to the start codon

2. Elongation: Amino acids are brought to mRNA by tRNAs & added 1-by-1 to growing chain

3. Termination: STOP codon in mRNA is recognized by the protein release factor & the translational apparatus comes apart, releasing the polypeptide.

* Describe the eukaryotic initiation phase of translation, specifically the roles of the start codon and initiation factors.

• There’s about 12 initiation factors

1. First initiation factor binds to the first tRNA (Met)

2. tRNA then binds the small ribosomal subunit

3. Other initiation factors bind the small subunit, creating the 43S preinitiation complex.

4. Complex recognizes the 5’ cap and binds the 5’ end of mRNA

5. Preinitiation complex scans the mRNA sequence for the start codon

6. Nucleic acid sequences surrounding the start codon help tRNA binding

7. After the initiator tRNA is base paired with the start codon, the large subunit joins with GTP hydrolysis

8. Most initiation factors are removed & translation can continue

Overall role of initiation factors: they help assemble the translation machinery. They guide the small ribosomal subunit to the mRNA, stabilize the complex, and ensure accurate start-site selection.

* What are the three cyclical steps that repeat during the eukaryotic elongation phase of translation, and what do elongation factors do?

1. Aminoacyl tRNA binds to the A-site of the ribosome, bringing the new amino acid into position.

   • It’s escorted by the elongation factor bound to GTP

   • When tRNA binds to mRNA, GTP is hydrolyzed & elongation factor is released & recycled

2. Peptide bond is formed between the terminal amino acid on the polypeptide chain & the new amino acid (in A-site). Bond between tRNA & amino acid in P-site is broken.

3. mRNA advances by 3 nucleotides. tRNA with the polypeptide chain moves to the P-site, the empty tRNA moves to the E-site & is released.

* Describe the general process of termination during eukaryotic translation, and what do the stop codon & release factors do?

1. Stop codons in mRNA are recognized by proteins called release factors, not tRNA

   • Release factors bind in A-site & terminate translation by releasing completed polypeptide

2. Two hydrolysis reactions:

   • Hydrolysis of GTP

   • Hydrolysis of last bond between tRNA & polypeptide

Most mRNAs are read by many ribosomes simultaneously, called a ________ or _______.

polyribosome, polysome

* Q: Kanamycin is an antibiotic that binds to bacterial ribosomes and allows tRNA with any anticodon to bind in the A site. What effect does kanamycin therefore have on bacterial translation?

It cause the incorporation of incorrect amino acids into a polypeptide.

1. Where does most polypeptide synthesis take place?

2. What are the 2 pathways after translation begins for routing protein products?

2. In the cytosol

3. Cotranslational import, posttranslational import

* What does protein processing or modification post-translationally do?

Changes protein function, can help sort proteins, etc.

1. * What type of signals from protein modification determine where proteins can end up in cells?

2. Where do they end up?

1. Localization signals

   • Ex: ER-retention tag, Nuclear export signals (NES), Nuclear import signals (NLS)

2. Endomembrane system, cytosol, mitochondria, chloroplasts, peroxisomes, nucleus

* What are the three types of single nucleotide substitutions (point mutations)? Describe their effect on the protein produced from a coding gene.

1. Missense: a nucleotide change that alters the amino acid specified by the codon.

2. Silent: a nucleotide change that does not change the amino acid, usually because of redundancy in the genetic code.

3. Nonsense: A nucleotide change that converts a codon into a stop codon, causing premature termination of translation.

* Eukaryotic gene expression (how much protein is being produced) is regulated at what 5 main levels?

1. Genome

   • DNA packaging (euchromatin & heterochromatin)

   • Epigenetic regulation: “histone code” (methylation & acetylation)

2. Transcription

   • Distal elements (enhancers, silencers, insulators)

   • Transcription factors (activators, repressors)

3. RNA Processing & Nuclear Transport

   • RNA splicing

4. Translation

   • Initiation factors

5. Post-translation

   • Protein folding/assembly, modifications, cleavage, import to organelles

* What is the difference between heterochromatin & euchromatin, & what is each’s effect on a gene’s expression?

1. Heterochromatin: chromatin is tightly packaged/compact, less access to transcription factors and RNA Pol II, inactive genes

   • Shows up as darkly-stained DNA in images

2. Euchromatin: DNA is loosely packaged, more access to transcription factors and RNA Pol II, active genes

   • Shows up as lighter-stained DNA in images

Contrast facilitative heterochromatin and constitutive heterochromatin.

• Facilitative heterochromatin: can be converted to euchromatin & vice versa

• Constitutive heterochromatin: permanently compacted, serves structural functions (centromeres, telomeres)

* What is the histone code?

A set/collection of post-translational modifications of histone tails in nucleosomes.

* What do specific modifications to histone proteins do?

Signals other factors, such as chromatin remodeling proteins, in the cell to “turn on and off” genes by changing the level of packaging of chromatin, allowing easier or more difficult access to DNA by transcription machinery.

* What are examples of cell type-specific transcription factors and how are they important in addition to general transcription factors to influencing a gene’s expression level (level of transcription)?

• Activators and repressors

• All cells have general transcription factors (GTFs) that are required for basal transcription, but the differences in gene expression between cell types come from cell type–specific transcription factors like activators & repressors.

• They increase or decrease transcription of certain genes based on what that specific cell needs.

* Differentiate between these 3 distal control elements & their effect on gene expression:

1. Enhancers

2. Silencers

3. Insulators

1. When activator transcription factors bind enhancers: affects the promoter to increase transcription

2. When repressor transcription factors bind silencers: affects the promoter to decrease transcription

3. Insulators: block an enhancer to prevent the transcription of an adjacent gene

* True or False: Differences in regulatory transcription factors in different cell types contribute greatly to differentiation of cells from one another, by turning on and off different genes.

True, activation or repression of certain genes can allow for specialization or differentiation of cells

* What are the characteristics of viruses?

• Viruses are incapable of free living

• They consist of nucleic acid & protein & sometimes lipid bilayer envelope

• * Viruses need to adapt to their host cell environment to properly survive & replicate themselves

• Use the host’s synthetic machinery to produce more virus particles

* What are the general steps of HIV’s life cycle within human cells?

1. Entering the host cell - fusion of viral membrane with cell’s membrane then releases core into cytoplasm

2. Reverse-transcribing its single stranded RNA genome into double stranded DNA
   (dsDNA) while being transported through cytoplasm by dynein along MTs

3. Nuclear import (nuclear pore complex proteins) then integration of its dsDNA into the host cell chromosome (using integrase)

4. Transcribing new RNA genomes and translating viral proteins in the host cell

5. Assembling and releasing new viruses from the host cell

6. Maturation of HIV virus

What are the main protein components of HIV?

1. Envelope (Env): directly binds to receptor on target cell

2. Capsid (CA): protects important viral RNA and proteins inside the core

3. Reverse transcriptase (RT): Uses single-stranded RNA genome as a template to make ds DNA genome, ready to integrate into the host chromosome

4. Integrase (IN): catalyzes the cleavage of the host chromosome & insertion of the viral dsDNA gene

* Which steps of the HIV life cycle require specific host cell factors?

1. Entry of cell mediated by binding of virus to specific receptor on cell’s plasma membrane, membrane fusion.

2. Trafficking of virus core along microtubules to reach nuclear envelope.

3. Associating with nucleopore proteins and passaging through the pore into nucleus.

4. Once viral genome integrated, using host transcription machinery to make viral
   RNA, host proteins to export RNAs to cytoplasm, and host translation machinery
   to make viral proteins.

5. Assembling new viruses at host cell plasma membrane, and using host cell factors to pinch off a piece of the host plasma membrane which now enclose new HIV viruses which can mature and infect new cells.