Genetics- MT 2- Lect 5 (09/30/2026)

Does DNA replication need a promoter

NOOO it has origins of rep and primase

What are basal and transcription factors

idk she skimmed it if u dont know the answer later on search it up

they recruit the RNA polymerase and the DNA polymerase

How is the template strand read

3’-5’ direction

What are the diff termination signals

• Rho-Independent (Intrinsic) Termination

• Rho-Dependent Termination:

Promoter are upstream or downstream of genes

Upstream in both pro and eu

What are promoters

Docking sites for transcription factors to recruit RNA Polymerase

When does transcription start?

After RNA polymerase binds

Enhancer sequences can be far upstream and they

impact the timing and level of transcription

Can the enhancers do anything without activators

Usually no

What are the key features in prokaryote promoters

-10 and -35 Regions (Prokaryotes)

• -35 region: DNA sequence located approximately 35 nucleotides upstream of the transcription start site. Helps RNA polymerase recognize the promoter.

• -10 region: DNA sequence located approximately 10 nucleotides upstream. Helps RNA polymerase bind and unwind the DNA.

• +1 position: The first nucleotide that gets transcribed into RNA.

What are the key features in eukaryote promoters

TATA box- at the -25 position important area for the transcription factors and basal to assemble which will then recruit our RNA polymerase

What is the +1

denotes where we will start synthesizing the transcript

Enhancers are x of genes

Upstream but also downstream

What are enhancers

docking sites for transcription factors to recruit RNA polymerase

RNA-like strand AKA

coding strand and non-template strand 5’-3’

Simultaneous transcription

A gene can be transcribed simultaneously by several RNA polymerases

Only the x strand is used to make mRNA

Template DNA

What way is the template DNA strand read

3’-5’ and copies via complementarity to create mRNA 5’-3’

Coding stand is complementary to…

the template stand but the T’s become u’s

Which end receives the nucleotide cap as its modification

5’- A modified guanine nucleotide with extra phosphate to help stabilize and help with recognition for ribosomes

Which end receives the 3’ end gets poly-A tail

50-250 Adenine nucleotides- both help protect mRNA from hydrolytic enzymes and nucleases and appear to facilitate export of mRNA so this happens before it is sent into the cytoplasm

Splicing

exons stay introns(intervening) leave

Random fact about restriction enzymes

All restriction enzymes are nucleases, but not all nucleases are restriction enzymes.

5’ UTR and 3’UTR

Untranslated regions

Are all expns translated

No but they ate all in the final mRNA strand

Splicosomes

Recognize splice sites- has catalytic function so proteins and rna can have catalytic function

Pre-mRNA

aka primary transcripts which then gets turned into mature RNA

How for spliceosomes know what to remove

specific nucleotide sequences in the pre-mRNA that mark where introns begin and end- there is a 5’ splice site and a 3’ splice site

How does the ribosome scan

5’-3’

What sets the reading frame

finding the start codon

Alternate splicing breaks the rule of one gene is equal to one product. Why?

Some genes can encode for more than one kind of polypeptide depending on exons- 100,00 proteins form 28,000 genes

Do prokaryotes have splicing

Yes but a little less

In prokaryotes can translation of mRNA begin before transcription is finished

yes which is why we can make polyribosomes where multiple ribosimes can translate a single mrna simoltaneously

Where else do we have translation

Mitochondria, chloroplast, Rough ER

look into dna methylation and aceltelation and lysene if she did not explain yet

What are the two things that need to match up of mRNA to get translated

tRNA needs the correct amino acid attached

tRNA anticodon must match the mRNA codon

also remember that we need a mechanism to uncompact the dna

How does the ribosome find aug

recognises 5’cap and keeps scanning till it finds aug but only for eukaryotes, for prokaryotes there is smt called Shine-Dalgarno sequence usually upstream of AUG as Prokaryotic mRNAs generally do not have a 5′ cap.

Anti codon is—— to the codon

complementary and antiparallel association so if mRNA is 5’ to 3’ then anti codon is 3’ -5’

How many common amino acids

20

Wobble effect

3rd nucleotide pairing is loose

trna have t or u

u

where do we have the wobble base

5’ end of trna

what is diff in each amino acids

R side chain

What site fills first

P and amino acids are connedcted from n-terminal to c-terminal

incoming tRNAs enter the A site, but the very FIRST tRNA

enters the P site instead

Where is the shortest polypeptide in the poly ribosomes

5’ end so moret below

what do scientists use it to figure out whether two mutations are in the SAME gene or DIFFERENT genes

complementation

What does studying mutant alleles allow?

Mapping of chromosomes

EX of studying mutant alleles

Eye color in Drosophila, drosophila have many diff phenotypes as thay have many shades of red- mutations cause them to be white

Complementation

• Different genes mutated in the parents → Complementation → Normal offspring.

• Same gene mutated in both parents → No complementation → Mutant offspring.

Mutations that do/do not complement each other are in a complementation group

Do Not

If wild type phenotype is restored

You have complementation

Complementation table

Group the negatives they will tell you which genes are on the same gene

How to think about it

Can you ever get x with y and have it positive (complement) No? then it is in the same complementation group and in the same gene

Complementation shows that

Mutations can be in diff genes or the same genes

If crossing over occurs between two mutations can wild type phenotype be restored

Yes but a very large sample size

Can a gene have crossing over within it

Yes and that could allow all the mutants to be isolated to one chromosome and the other does not have any mutations

Seymour Benzer (1950s)

T4 bacteriophage infects E-coli making a lot more viruses

Why was Seymour Benzer’s experiment important

It showed that recombination can happen between different positions WITHIN the same gene, not just between different genes.

Bacteriophage

virus that infects bacteria

Plaques

Clearings in a lawn of bacteria where 1 T4 has entered and spread to adjacent cells causing lysis of the e coli so they dead which leaves a zone without bacterial growth- the whole plate is bacteria that is not affected by the virus the specific circles we see are plaques infected with the virus

Wild type vs Mutant phenotype in E.coli for the bacteriophage T4 experiment system

rll= rapid lysis

rll- are the mutants and that results in larger plaques with a bigger better circular shape

rll+ are the wild type

Explain the bacteriophage T4 Experimental systems based on mutant vs wild and specific type of e coli strain

E.coli strain B allows the reproduction of viruses for both mutant and WT but K only allows rll+ to reproduce

Each bacteriophage is

monoploid, meaning it only has one copy of each gene

How can we get recombination with only one copy?

Dual infection- simultaneously infect bacteria with two diff bacteriophages- both chromosomes are injected into the ecoliu so recombination can occur

T4 bacteriophage experiment setup

When infecting e.coli B is this a genetic screen or selection

• Answer: Genetic Screen

• Why: Both wild-type ($r^+$) and mutant ($rII$) phage strains can successfully infect, grow, and form plaques on E. coli B. Because both phenotypes survive, researchers must examine the individual plaques (distinguished by morphology) to identify mutants, which defines a screen rather than a selection

When infecting e.coli is this a genetic screen or selection

• Answer: Genetic Selection

• Why: Only wild-type ($r^+$) or recombinant wild-type phages can grow and form plaques on this strain. The rII mutants cannot grow on E. coli K($\lambda$). This conditions out the vast majority of the mutant population and allows only the rare desired recombinants to survive and form plaques, which defines a selection.

answer 2

What are the two outcomes after crossing the bacteriophage with an uncharacterized (a mutation whose exact location on the DNA is UNKNOWN) mutation and a known deletion

Wild type or no wild type

If no wild-type recombinants are produced…

the point mutation is likely INSIDE the deleted region or there are overlapping deletions

If wild-type recombinants are produced

the point mutation is OUTSIDE the deleted region on the other chromosome or the deletions are non-overlapping

Deletion Mapping

Allows you to figure out where mutations are because when the wild type can not be produced and is -/O that means that the location of that mutation is where the deletion is

What is the order

D E B A C

Recombination frequency formula- genetic distance

we multiply by 2 because the mutant viruses die on the plate- add m.u. at the end

how to get physical map recomb frequency from genetic recomb frequency

multiply m.u. with 113 base pairs ending unit is bp

Benzer’s Conclusions

1. Mutations can be single nucleotides\

2. A gene is a continuous linear sequence of nucleotide pairs

3. Nucleotide sequences composing two genes are seperate and distinct

Hotspots

Sites where mutation occurs spontaneously more often than other sites are called hotspots

Does complementation restore function or genetic sequence

complementation restores function, not genetic sequence

Benzer’s Complementation Experiement

• Complementation experiment: E. coli K was co-infected with two different rII− mutant phage and saw whether cells lysed (complementation) or not (no complementation)

• The purpose was to determine which mutations were in the same gene.

Benzer’s Recombination Experiment

Recombination experiment: E. coli B was co-infected with two phages with different mutations in the same gene. rII− mutants can grow in this host where genomes can recombine.

• Looked for whether progeny phages became rare rII+ recombinants able to grow on E. coli K.

Central Dogma of Biology

DNA inherited by a chromosome will dictate specific traits by serving as a template for protein sythesis

What affects phenotypes

Proteins

What leads to cell specialization

Gene expression- when they turn the genes on, if they turn it on and the level of genes and protein that is coming out of those transcribed genes- based on both transcription AND translation

Rll a and rll b vs Rll A1 and Rll A2

Rll A and Rll B are both types of rll- to see if the genes werecomplementary

Rll A1 and Rll A2 are both types of Rlla- used for seing if recomb occured

What is the difference in prokaryotes and eukaryotes with the mRNA

the rna transcripts is directly the mRNA in pro and has to be processed to become mRNA in eukaryotes

Transcription

RNA Polymerase- Pries the DNA strands apart and builds the RNA nucleotide strand

Having RNA polymerase removes the need for what

Primer (unlike DNA polymerase RNA polymerase can start a chain from scratch and no 3’end is needed) and helicase

RNA is ——- to the DNA template

Complementary

Is the newly transcribed RNA the same as the coding strand except the T becomes U?

Yes

tRNA structure

The long length is 3’ short length is 5’- anticodon antiparallel amino acists get attached to the 3’ ACC group

where are the epa sites

in the ribosomes- eject-polypeptide- acceptor

What is the reason we do not have just a ton of tRNAs

wobble position

polyribosomes

are only in bacteria multiple ribosomes can translate a single mrna while multiple rna polymerase can transcribe as well

simultaneous transcription