DNA/RNA.lecture



BIOL V04 COVID-19 Emergency Lecture Alternative © 2020 copyright Marta D. de Jesus

BIOL V04 Lecture: From Gene to Protein (Campbell Biology - Ch 17)

I. How does DNA affect phenotype?

Slide 2

A. Archibald Garrod (1909)

1. physician studying “inborn errors of metabolism”

2. discovered alkaptonuria (urine turns black) followed a Mendelian pattern of inheritance

Optional reference:

Slide 3

B. George Beadle & Edward Tatum (Nobel Prize Physiology/Medicine 1958) - classic experiment

“1 gene - 1 enzyme” (this saying is now modified)

Overview:



Fairly good explanation of the nutrition experiment, but the organisms were Neurospora (a fungus) not E. coli (a bacterium)



Optional reference:

Slide 4

C. Overview: information flow and gene expression within a cell

1. prokaryotic version: both transcription and translation occur in the cytoplasm

2. eukaryotic version

a. more compartments

b. more process complexity

II. Transcription = synthesizing RNA

Slide 5

A. Structure

1. monomers = nucleotides; differences from those used in DNA

a. different sugar

b. 1 different nitrogenous base (other 3 remain the same as what’s found in DNA)

Note! ATP/ADP/AMP used in metabolism are ribonucleotides

2. polymer is RNA

a. (usually) single-stranded

b. short (relative to DNA; usually contains 1 gene’s worth of information); DNA’s usually waay longer

B. When does this happen in the life of a cell?

(Recall Ch 12!)

Slide 6

C. 3 main kinds of RNA

1. messenger RNAs (mRNA) - usually has a ribbon-like shape

2. Other 2 types of RNA - protein-producing machinery

Slide 7

a. transfer RNAs (tRNA): 3 different ways to think about its shape

1) built to carry specific amino acids (see amino acid attachment site at the 3’ end)

2) and H-bond to a specific codon on an mRNA at the ribosome with its anti- codon

Slide 8

b. ribosomal RNAs (rRNA): complex 3D structures

1) 2 subunits: large and small

2) specific pockets for tRNAs and grooves for reading a mRNA and constructing (and releasing) a polypeptide

Slide 9 - do not memorize the components shown on this slide

3) ribosomal subunits are made of rRNAs and proteins

prokaryotic 70S ribosomes are smaller and the subunits are smaller

than

eukaryotic 80S ribosomes (and ribosomal subunits)

-> but there are other kinds of RNAs (1 more kind in Slide 16; others will be discussed in another chapter)

Slide 10

D. How is it made? Transcription

1. What does that word refer to? transcribing music/ courtroom transcript = very faithful copy in the original “language” (in this case, in nucleotides)

2. similar-looking

a. to DNA replication process (in some respects)

b. the DNA information (faithful copy)

3. RNA polymerase: enzyme that can make RNA

a. can only add nucleotides to the 3’ end of an RNA

critical fact for understanding: RNA (like DNA) is only made and read 5’ -> 3’ direction

b. searches for special DNA sequence locations:

1) promoter - signals the start of transcription (“the gene starts near here”)

2) termination sequence - signals the end of transcription

3) transcription unit = the DNA between those 2 places

c. biosynthesis - constructing a polymer

like with DNA, the nucleotides provide the energy (come in as triphosphates)

4. Steps of the process:

a. Initiation starts at a promoter, which includes:

1) initiator site = location where RNA synthesis actually starts

2) some upstream (before the gene) nucleotides

3) RNA polymerase starts the unwinding of local region (1 turn of DNA)

b. Elongation

adds in new RNA nucleotides according to complementary base pairing, reading template strand (only 1 side) in 5’ -> 3’ direction

c. Termination & release of RNA transcript

termination site containing termination sequence (on the DNA) tells RNA polymerase to let go of the DNA

Note: not related to terminator codons!





(through 2:50)

More realistic view





Slide 11

d. 1 chromosome can have:

1)

2)

Slide 12

E. major differences between prokaryotes & eukaryotes

1. in prokaryotes

a.

b.

c.

d.

Slide 13

2. in eukaryotes

a.

b.

c.

Slide 14

d. transcription start (initiation)

1) on DNA, influencing initiation within promotor: TATA box: at ~25-35 bp upstream of start point

not shown on slide

2) on DNA, outside of promotor

- upstream promotor elements (UPEs): starting at ~80-100 bp upstream

- enhancers = DNA sequences up or downstream that help

- only functional in some cells, though present in all

- can be very long distances away

- can be inverted & still functional

- work through proteins called activators

3) transcription factors: proteins that bind to DNA and affect transcription

- can be activators (help transcription happen)

- can be repressors (shut off transcription)



Note: your textbook has even more details than this, but you are only responsible for the ones mentioned in this document.

Slide 15

e. post-transcriptional modifications of pre-mRNA = RNA processing

1) capping at 5’ end with?

Why? (What does this do functionally?)

2) tailing at 3’ end with?

Why?

Slide 16

3) removal of introns and splicing of exons

- exons code for?

- introns don’t code for?

- process involves

- small nuclear RNAs (snRNAs)

- small nuclear riboproteins (snRNPs)

- together these make spliceosomes which do?

(are an example of ribozymes)



More realistic



Slide 17

f. Why are euk. genes like this?

One explanation: allow for alternative splicing -> can get more than 1 mRNA from a eukaryotic gene, thus more than 1 protein.

Slide 18

III. Translation = making a polypeptide (how is the mRNA is read/used)

A. foreign language analogy: moving from nucleotide-based language to amino acid-based one

Slide 18

B. what is it saying? The Universal Genetic Code

1. codons = patterns of 3 (triplet) nucleotides on mRNA that code for a specific amino acid or say “Stop”. Only one to memorize for BIOL V04 is the one that can mean “Start” (it also means something else).

a. Why so many codons?

1) How many nucleotides are used to make DNA or RNA?

2) How many amino acids are used to make proteins?

3) If one uses a 1-nucleotide-based code, how many amino acids can be specified?

4) If one uses a 2-nucleotide-based code, how many amino acids can be specified?

5) If one uses a 3-nucleotide-based code, how many amino acids can be specified?



6) special codons: 3 Stop/Terminator codons (tell the ribosome its done working)

1 Start codon {also says “put in Methionine (Met) as the next amino acid”}

b. since there are “extras” the code is redundant/degenerate (= some amino acids have more than 1 code), but not ambiguous (= code never switches to saying can put in another different amino acid)

c. universal = the same for all organisms

1) you use it, a dog does, your E. coli do, etc.

2) makes genetic engineering quite possible

e.g.: human proteins now made as pharmaceuticals by other organisms

Slide 19

d. originally read by making synthetic mRNAs & seeing what was made

1) 1961: Marshall Nirenberg & JH Matthaei deciphered 1st codon UUU => told ribosomes to put in Phenylalanine (Phe)

2) Nobel Prize 1968: Nirenberg, Khorana & Holley for the entire code & how it’s used

Optional: Story about that time:

Slide 20 & 21

C. How is a protein built?

1. Takes ribosomes = protein factories

a. made of large & small subunits

these are made of?

b. have 3 sites (pockets) for tRNAs

1) P site = full name? function?

2) A site = full name? function?

3) E site = full name? function?

Note: some older figures and animations do not show the E site; it was more recently discovered.

c. perform enzymatic (catalytic) activities

another example of a ribozyme

Optional: ribosome structure:

2. Takes tRNAs carrying?

Slide 22

a. How do tRNAs get their amino acid to carry? charging the tRNAs

1) requires enzymes: aminoacyl-tRNA synthetases join tRNAs and amino acids

2) requires energy (ATP)

Slide 23

b. tRNA structure

1) short & t-shaped (when flat)

2) anticodon loop for recognition of codons of mRNA

3) should be 61, but only ~45

- wobble = anticodons are less specific than codons; allow some other H bonding matches in the 3rd position

3. mRNA to tell the ribosome how to build the protein

4. biosynthesis: making a polymer so requires energy; this time its GTP

Slide 24

D. Steps:

1. Initiation

a. 3 participants meet first

1)

2)

3)

b. then 4 one:

-> initiation complex

Slide 25

2. polypeptide Elongation

a. codon recognition

b. peptide bond formation

c. translocation

d. repeat

Slide 26

3. polypeptide Termination

Stop codon

Release factor



More realistic





E. in prokaryotes - can see polyribosomes/polysomes reading 1 mRNA



F. proteins fold into their final shape by interacting with themselves and their environment

may require help: chaperone proteins while they are being made

G. post-translational modifications

1. amino acids in a polypeptide can be modified (e.g.: glycosylation)

Slide 27

2. signal sequences on the protein can tell the cell where it goes next (analogous to zip codes)

a. proteins for the RER (Gunter Blobel - 1999 Nobel Prize in Physiology)

1) signal peptide/sequence is first part of polypeptide to be made

2) signal-recognition particle (SRP)

- binds to the signal peptide

- stops further translation until

3) SRP binds to a receptor protein on the RER

4) SRP is released & the growing peptide is pushed into interior of RER through a channel and translation continues

5) when polypeptide is completed, signal sequence is removed (if protein is to be secreted)

Slide 28

3. further post-translational modification can occur

editing the polypeptide eg: processing of insulin

IV. How accurate do all these processes have to be? Try to be extremely accurate but still changes can happen

Slide 29

A. mutation = any change to the DNA

can be caused by many things: chemicals, radiation, mistakes (spontaneous), etc.

B. point mutation = 1 (nitrogenous) base change

1. base-pair substitution

a. silent = neutral

no change to polypeptide

(how does this happen?)

b. missense = 1 amino acid to another; can be unnoticeable (how?) to dramatic (how?) eg: sickle-cell anemia (dramatic)

c. nonsense = amino acid -> (premature) stop; can be unnoticeable (how?) to dramatic (how?)

2. frameshift mutation = move reading frame (can be larger than point)

a. base-pair insertion

b. base-pair deletion

C. larger DNA changes

1. chromosomal: deletion, insertion, translocation, inversion, etc. (see Ch 15)

Slide 30

2. transposon a.k.a. “jumping genes” = pieces of DNA that can move into/out of place

(Barbara McClintock 1983 Nobel Prize in Physiology)

Slide 31

3. DNA viruses and retroviruses: can insert genetic material into an organism & sometimes its chromosomes e.g.: HIV (a retrovirus)

Slide 32

D. is mutation always bad? can be situational

e.g.: sickle-cell trait

in USA -> threatens health of the people who have it

area of the world with Plasmodium falciparum-based malaria -> heterozygotes are protected

Skip: E. How to test chemicals for mutation-causing ability

1. for mutagens

2. for carcinogens

Other helpful resources:

NDSU Virtual Cell: Transcription



NDSU Virtual Cell: Translation



Khan Academy: Transcription



Khan Academy: Translation



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