bio 275

KCC with Matthew exam 2

3 main topics for ch 7

• DNA to RNA

• RNA to protein

• RNA and origins of life

why is transcription called such

• DNA to RNA

• same language but different form

 Why is translation called such

•  RNA (4 nucleic acid)  to protein (20 amino acids)

• Different language

 How would you define a gene?

Region of DNA required for synthesis of functional product

Are genes expressed at different rates, would this affect cell processes?

•  Yes

• Low level gives small signal, higher level gives more signal

Could you easily rattle off 4 differences between DNA and RNA?

•  DNA sugar deoxyribose, C-G 3 hydrogen bonds, double stranded, read 3’ → 5’

• RNA sugars ribose, A-U 2 hydrogen bonds, single stranded, formed 5’ → 3’

DNA

sugar deoxyribose, C-G 3 hydrogen bonds, double stranded, read 3’ → 5’

RNA

sugars ribose, A-U 2 hydrogen bonds, single stranded, formed 5’ → 3’

Do all pairings follow the Watson-Crick base pair rules?

no

 Which is the template strand, the top or the bottom?

bottom strand

Which molecule can adopt more configurations, ds DNA or ssRNA

 ssRNA

What is the coding/sense strand of DNA?

 Serves as a template for synthesis of a complementary RNA molecule

 What is the template/non-coding/antisense strand of DNA

Provides the complementary sequence that guides the synthesis of a complementary RNA molecule during transcription

Who is the key enzyme for transcription, and where does the energy come from?

 RNA polymerase which comes from cleavage of high energy phosphate bonds

Which 3 RNA “types” are utilized in gene expression?

• Messenger RNA 

• Transfer RNA 

• Ribosomal RNA

Can you name 4 items used in bacterial transcription?

• Promoter: promotes gene expression. Directly upstream of start 

• +1 position: start site 

• Terminator: the box is flipped 180 and creates a hairpin which makes the polymerase to fall off and stop 

• DNA template

• RNA polymerase

• If flipped 180 then the transcription occurs in opposite strand in opposite direction

What is the RNA polymerase used in eukaryotic cells?

 RNA polymerase I, II, III

 In bacteria, where are 2 motifs that regulate the start site of transcription?

•  -10 promoter element 

• -35 promoter element

 Why does the tail end of a bacterial gene often have a palindromic sequence?

It is sequences of DNA or RNA that read the same backward and forward

Are different DNA polymerases used in human cells for different processes?  Are different RNA polymerases used in the cell for different processes.

yes

What is the TBP, and where does it bind?

Tata binding protein, binds to the TATA box 5’-TATAAAA-3’

 Can you explain the 5’ cap, and the advantages of having one?

•  It’s an inverted G that contains an additional methyl group 

  • Limited to eukaryotes added after ~25 bases are transcribed

• Advantages 

  • Limits premature mRNA degradation 

  • Supports porper/regulated degredation 

  • Enhances translation, splicing, and export

Can you explain some advantages of the 3’ polyadenylation?

 Regulates mRNA longevity/turnover, also enhances nuclear export

Do eukaryotic genes contain introns, what are these and why might they exist?

Yes, they contain key sequences that direct the spliceosome to dock and excise it

What is a spliceosome composed of, and which component actually performs catalysis?

Spliceosome is a Machine that splices introns and joins together exons. Made up of complex of snRNPs (small nuclear ribonicleoproteins). It is a ribozyme and the RNA portions of the complex are responsible for catalysis

Are genes transcribed all throughout the genome, akin to starting millions of little fires concurrently?  Or, are genes threaded to preassembled factories to allow for transcriptional hot spots?

Genes are preassembled to allow for transcriptional hot spots

What is meant by the statement that the genetic code is “redundant but not ambiguous?”

 Lots of repeating codons but it always has the same outcome

Can you explain the “wobble effect?”

First 2 letters od codon are most important the 3rd not so much

How many sense and antisense codons are there?

61 sense codons, 3 antisense codons

What do we mean in saying the “genetic code is universal?”

• Cut between and paste and move genes between species 

  • Bad because virus can use out machinery to translate and transcript

What are the 2 most important portions of a tRNA molecule?

• Anticodon 

• Aminoacyl head

Would you consider a ribosome an organelle?  What is a ribosome composed of?

 Composed of specific proteins and rRNA molecules

What is a polycistronic mRNA, why is it so nifty?

Multiple coding regions, code for multiple polypeptides

How is translation terminated?

Reaches one of the 3 stop codons. There no corresponding tRNA that would be complementary. Instead release factor comes in and binds and causes disengangement of whole complex

What is a proteasome, and why is this compartment useful?

Proteasome contains proteases. Breaks down proteins, regulated protein levels, remove damaged proteins

What are the 4 main sections of ch 4

• The Shape and structure of proteins

• How proteins work

• How proteins are controlled

• How proteins are studied

Briefly explain 6 functions or proteins within humans.

•  Enzymes 

• Structural

• Transport 

• Motor

  • miocin, actin 

• Storage

• Signal

• Receptor

• Transcription 

• Special purpose

  • Green fluorescent protein

You must be able to ascribe an amino acid to 1 of the chemistry groups, practice here?

• Tyr polar (tyrosine)

• Leu non polar (leucoine)

• Asp acid (aspartic acid)

• His base (histidine)

What are the 4 common electrostatic interactions that influence polypeptide shape?

• H bonds

• Electrostatic interactions (ie. ionic bonds)

• van der Waals attractions

• Hydrophobic forces

Polypeptides are typically held in high or low energy shapes?

 Low energy because less reactive and more stable

Can you denature and renature polypeptides, what do these experiments tell us?

Yes, the a.a. Sequence is enough to dictate proper folding

What are chaperone proteins, and why are they so useful?

• facilitate folding and not destroyed in the process (Assist other proteins in obtaining confirmation)

  • Bind to group and limit interactions

  • Short term interactions, non covalent

Are beta sheets rigid, do they provide internal structure to a polypeptide, and where would you find the side chains for the participating amino acids?

 They are ridgid and serve as a core for many proteins.

What are the downside of beta sheets, and in which situations could you find them?

 Can become insoluble and difficult to destroy, they may increase with time and promote diseases ex. alzhiemers , huntingtons, mad cow

What are the 4 levels of protein organization, can you explain and recognize each?

1. Primary structure 

   1. Popypeptid strand

2. Secondary structure 

   1. Helix and pleated sheet

3. Tertiary structure

   1. Helix and pleated sheet fold into 3D shape

4. Quaternary structure 

   1. 2 or more polypeptide complex’s together

 Given the nearly limitless possible configurations of a single polypeptide, provide 2 reasons why nature actually maintains only a modest set of protein chains.

•  Cells can only make a fraction of the possible chains because require stable well defined 3D confirmations 

• Natural selection shaped proteins to be stable and reliable

 So is it true that once nature finds a stable and useful structure (and its corresponding sequence), gene/protein families are expanded and drift into different uses (while still retaining very similar shapes)?

yes

Quick review – what is a domain, what is a subunit?

•  subunit: one polypeptide in larger structure 

• Domain: functional region of a larger chain

Which types of proteins are typically globular, and which types of proteins are typically fibrous?

• Enzymes are globular Ex. collagen

• Spanning proteins are fibrous Ex. elastin

Enzymes are responsible for nearly all chemical transformations within a cell, but why do we say “nearly?”

Typically bind one or few substrates

Which is your favorite enzyme class, why?

Phosphate because catalyzes the hydrolytic removal of a phosphate group from a molecule

What is the difference between a cofactor and a coenzyme?

• Cofactor is minerals 

• Coenzymes is vitamins

Explain how feedback regulation can shunt one branch of a synthesis pathway… but allow other branches to proceed.

•  Only shuts down one arm and other branches can proceed 

• Final pdt negatively regulates the initial synthesis step

Just by getting an early taste of biochemical pathways, does this encourage you to pursue a biochemical career?

no

 Why can’t the final pdt (of a pathway) bind (and compete with substrate) at the active site of an enzyme?

Because the thing is converted into something else with new shape and the molecule is different so it won’t work

What are the 3 typical amino acids that serve as targets for kinases?

 serine , threonine, or tyrosine

 What are scaffolds, are they composed of protein or RNA, and how do they relate to cellular tasks?

• Helps bind proteins with related functions proximally orienting them and making a favorable microenvironment. 

• Composed of polypeptide or RNA

Again, what are some advantages of cholesterol in animal membranes?

•  Cholesterol is used to control fluidity 

  • Stiffens the membrane making it less flexible and permeable

Do you also notice that transcription is the interplay of cis and trans factors?

yes

What are the 2 classes of proteins required to aid in transcription?  Also notice there is sequential binding and activation.

• Transcription factors

  • General and specific transcription factors

Did you notice that different transcriptional proteins have different roles, and some are influenced by other (upstream) events… so txn is a culmination of numerous information streams?  In a simple sense – txn go/no go is a combinatorial event.

yes

What is a primary transcript?

Primary transcript (Pre-mRNA) is the initial RNA molecule synthesized during transcription

What is a mature/processed transcript?

mature/processed transcript is the result of post transcriptional modifications which include splicing to remove introns

 What are 3 modifications done to eukaryotic mRNA?

3 modifications are splicing, 5’ cap, polyadenylation

How does a human cell know where to remove an intron, and what is the enzyme

•  Knows by recognizing the intron-exon boundaries through specific sequence elements found at the junctions 

• Spliceosome

 Alternative splicing is so very useful because?

•  Diversity of gene

• Tissue specific functions

• Efficient use if genetic information

Can just any old molecule leave the nucleus… or is there discrimination?

 Not any molecule can freely leave the nucleus. Nuclear export is tightly regulated

What items on mRNA facilitate it’s nuclear export?

Exportin, RNA processing, Ran-GTP

What is codon bias?

 Preference for certain codons that encode the same amino acid

What is a reading frame, can it be altered, can it overlap?

• Specific way of dividing a sequence of nucleotides into sets of consecutive, non-overlaping codons 

•  Viruses can have overlaping sequences

Are there more than 4 RNA letters, do you think there are more than 4 letters of DNA?

 Yes because of synthetic bases

Which enzyme reloads the tRNAs, where does it this happen, and how accurate must this enzyme be… and why?

•  aminoacyl-tRNA synthetase

• Happens inside the cytoplasm of the cell 

• It has to be very accurate because if not it can cause misfold or nonfunctional proteins 

 

What are the 3 regions of the ribosome holoenzyme?  What is each region responsible for?

•  Small subunit 

  • Decoding mRNA sequence

• Large subunit 

  • Catalyzing the formation of peptide bonda between amino acids 

• Exit tunnel and E site

  • Growing polypeptide chain exits the ribosome as it is synthesized

What is the direction of polypeptide synthesis, which end emerges out the top of the ribosome?

• Polypeptide synthesized from the N(amino) to C (carboxyl) direction 

• The newly added amino acid becomes the N terminus of the growing polypeptide

Besides the ribosome, are there other molecules needed to allow for the elongation of tln?

Yes ex. GTP, aminoacyl-tRNAs, tRNA and mRNA

Within the catalytic site of the ribosome, who holds the participating amino acid members in close, secure proximity… and performs catalysis?

 Peptidyl transferase center

After synthesis, what amino acid is always found first, although it may be cleaved later?

 Methionine “Met” or “M”

What does a Shine-Dalgarno sequence dictate

prokaryotic translation initiation and helps position the ribosomal on the mRNA. Ribosome localization sequence

what foes a Kozak sequence dictate

eukaryotic translation initiation and helps identify the correct start codon

 What are polyribosomes, what is txn-tln coupling, why are these things so special?

•  Clusters of ribosomes that are simultaneously translating the same mRNA molecule 

• Ribosomes can start translating an mRNA molecule while it is still being transcribes by RNA polymerase 

• Specialized structures that significantly enhance the efficiency of protein synthesis in cells

is there an amplification of signal during txn?

•  Amplification during txn

  • Mainly eukaryotic, Transcription of a single gene into a large number of mRNA molecules

 Is there an amplification during tln?

• Amplification of tln 

  • Mainly prokaryotic, Multiple ribosomes simultaneously translate the same mRNA molecule

Does protein shape matter?  How about post-translational modifications?  And location? And half-life?

•  Yes, it determines its ability to interact with other molecules 

• Yes, post-translational modifications can alter a protein's structure, stability, activity 

• Yes, proteins are targeted to specific organelles based on their localization signals 

• Yes, regulates protein abundance and turnover

What is the small molecule used to tag a polypeptide for destruction?

 ubiquitin

Why do you think there are so many steps between DNA and polypeptide in a human cell?

Because it ensures precise control, regulation, and fidelity of protein synthesis

Once a polypeptide rolls off of a ribosome, is it immediately ready for function?  Can you list some events that proteins undergo after tln?

• no, not ready immediately 

•  Protein folding 

• Localization 

• Subunit assembly

 List out various pieces of evidence that indicate RNA was the initial molecule coding life.

• RNA can store genetic information 

• RNA can catalyze reactions

• RNA self replication 

• RNA’s role in protein synthesis 

• RNA is an intermediary

Is the polypeptide backbone exciting or flexible – explain.

•  Not necessarily exciting, but relatively flexible 

  • The single bonds allow free rotation around the axis of the bond

What is the covalent bond that can form between select variable groups, what amino acid is involved in this “molecular staple?”

• Disulfide bond 

• cysteine

Why is so much money and effort put into computer modeling of proteins?

 Accelerates research because proteins have a wide range of application across medical and industrial domains

What are the stable, default structures that form spontaneously from the polypeptide backbone?

Secondary structures

With regards to an alpha helix, are the side chains internal or external?  Does the surrounding milieu matter, and do the side chain moieties matter?

• In alpha helix the side chains are oriented externally 

• The The side chain and surrounding milieu can influence the stability and properties of the alpha helix

What helps to hold coiled-coils coil together, are they amphipathic in some way?

Hydrophobic interactions and they are amphipathic

What are the names of modular regions within the larger polypeptide?  Are these connected directly to one another or is there a flexible tether/spacer between these regions?

•  Domains 

• Domains are connected to each other within a protein by flexible linkers or tethers

If you were given an amino acid sequence of a protein domain, and saw it was similar to other domains of known shape and function… could you make predictions?  

You could predict the function, structure, binding patterns

 Is a polypeptide a protein, is a protein a polypeptide?  Can you explain these differences?

 no, but polypeptides are fundamental components of proteins

 Can polypeptides join to make larger, repeated structures, can you name a few?

Yes, coiled-coils, collagen, fibrous proteins

Often the extracellular milieu is harsher than the internal compartment, therefore which type of bond it often found in proteins that function there (in the former, not the latter)?

 Disulfide bonds

I bind, therefore I am (a protein).  So what types of interactions typically allow different proteins to engage?

•  Non-covalent interactions 

• Covalent bonds

• electrostatic interactions

• Binding pockets and active sites 

• Multiple interaction sites

Are binding sites composed of precise amino acids arranged in precise ways?  Can distal binding sites influence the binding of proper ligands at alternate locations?

yes