Genetics Wxam 3

The three modes of DNA replication

conservative, semiconservative, Dispersive

Where does DNA replication begin?

Origin of Replication

How many origins are in prokaryotes and eukaryotes

1 in Pro, many in Eu.

Replication Initiation: proteins and function

**Initiator protein**: binds to origin and unwinds it

**DNA helicase**: further unwinds DNA

**Single strand binding proteins**: prevents DNA from winding back together

**DNA Gyrase:** lowers torsional strain by cutting DNA and join DNA back together

Bidirectional

Replication of DNA in an origin occurs in opposite directions

\# of forks per origin

2

Replication elongation: proteins and function

**DNA polymerase**__:__ builds DNA by attaching nucleotides to free 3’ -OH

**Primase**: Adds complementary RNA primer made of ribonucleotides which generates 3’ OH

**β-sliding clamp**: keeps DNA polymerase in place

**Clamp Loader**: loads beta sliding clamp on DNA

Direction of DNA polymerase

5’ to 3’

Leading strand directions

Template: 3’ to 5’ and replicates 5’ to 3’

Lagging strand directions

Template: 5’ to 3’ and replicates 5’ to 3’

How does DNA polymerase ensure that DNA is replicated correctly

**Nucleotide Selection**: check if the added nucleotide has proper hydrogen bonding.

**Proof Reading**: Detects is base pair is mismatched then uses 3’ to 5’ exonuclease activity to replace the base pair.

Termination of Replication

Eukaryotic: Two replication forks meet

Prokaryotic: Termination sequences that end replication Termination sequence

Termination: proteins and function

**RNAse H**: Removes RNA primer

**DNA ligase**: bonds fragments of DNA

**Topoisomerase**: uncoils precatenase

Precatenase

Newly synthesized DNA coils with parent

Catenase

Interlocking of 2 DNA molecules

Decatenation

The process of freeing up the daughter DNA molecules that are interlocked so that they can be distributed to daughter cells upon cell division

Telomere

Repetitive sequences at the ends of DNA that are lost during replication in place of important genetic information.

Enzyme that helps extend telomeres and how it does that

Telomerase: places a RNA template that extends DNA to which primers can attach and fill in a gap from the End Replication Problem

Features of telomerase

RNA dependent DNA polymerase

Centromere:

middle point where chromatids meet, Heterochromatin

Telomere (the location):

ends of C’some, heterochromatin

p arm

Short arm of C’some

q arm

Long arm of C’some

Non coding region def and examples

Do not code for RNA or proteins,

EX: intron, satellite DNA, regulatory sequence, repetitive DNA (telomeres, Interspersed repeats, Tandem repeats)

Locus

Location of a gene

Promoter

Sequence that aids in RNA synthesis

Coding sequence

Sequence that codes for an RNA molecule

Terminator

Signals the end of transcription

Rules: +1

DNA sequence where transcription begins

Rules: Negative numbers

sequence prior to start site

Rules: Positive numbers

sequence after to start site

Upstream

left of the sequence of interest, can be other things besides the coding sequence

Downstream

right of the sequence of interest

Central Dogma

D > D: Replication

D > R: Transcription

R > P: Translation

Reverse Transcription means?

RNA > DNA

Enzyme that mediates reverse transcription and when does reverse transcription occur?

Reversetransciptase, occurs when host cells are infected by viruses

Bacterial RNA polymerase

Function:

Composition:

Special:

Function: Preforms transcription

Composition: β, α, β᾽, ω, σ

Special: holoenzyme without σ factor

σ factor

allows RNA polymerase to identify promoter

Initiation of Transcription: in Pro.

Sigma factor in RNA polymerase allows binding to promoter

Coding Strand

Strand that is not the template for the RNA polymerase but is exactly like what it produces but DNA instead of RNA.

Template Strand

Used as a template because its complementary strand is what we’re looking for.

RNA polymerase direction

5’ to 3’

Transcription elongation process in prokaryotes

occurs in transcription bubble with RNA continuously created in the 5’ to 3’ direction, DNA unwound at front rewound in back

Elongation: what kind of nucleotides are added in transcription

Riboneucleotides

Rho-dependant

Rho bind to rut site, RNA polymerase stalls at terminator sequence, rho catches up and uses helices activity or unwind the DNA RNA hybrid.

Rho-independent

Inverted Repeat forms hairpin structure which destabilizes A-U pairing.

Transcription in eukaryotes: How does DNA become accessible for transcription

Heterochromatin must be remodled to Euchromatin

RNA poly II

creates pre-mRNA

RNA polyermaerase II termination mechanism

uses protein like RAT1

mRNA

Function:

Function: has information to make a polypeptide

In Eukaryotes: pre-RNA is modified into mature to RNA

5’ cap def and what links it to the mRNA and what it does

Addition of 7-methyl guanine to 5’ end of mRNA, prevents degradation. triphosphate linkage. Increases mRNA stability

Poly A tail

Strand of A’s added to 3’ end of mRNA

Splicing

Removal of introns and joining of exons

Alternate splicing

picking and closing which exons to express which forms a different protein for each combination

Splicing is mediated by (protein)

spliceosome

RNA that mediates splicing

snRNA

structure formed by spliced introns

lariat

RNA splicing steps order: Branch point, 5’ splice site, 3’ splice site

5’ splice site, Branch point, 3’ splice site

Post transciptional processes for RNA

RNA splicing, 3’ cleavage and addition, addition of 5’ cap

rRNA

Building blocks of ribosomes

70s ribosomes are found in?

Prokaryotes

80s ribosomes are found in?

Eukaryotes

70s ribosome composition

Subunits: 50s and 30s, rRNA: 23s and 16s

80s ribosome composition

Subunits: 60s and 40s, rRNA: 28s and 18s

tRNA must be ____ to form ____________

processed, mature tRNA

rRNA must be ______ to form _______

processed, mature rRNA

rRNA is processed by ___________

snoRNA and snoRNP’s

siRNA and miRNA length

21-25

siRNA snd miRNA bind with proteins to form ____________

RNA-induced silencing complex (RISC)

siRNA origin and creation

Foreign double stranded RNA which is cut into pieces by dicer making many siRNA

siRNA function

Perfect binding to mRNA causing degradation

miRNA origin and creation

RNA transcribed by a gene, pri-mRNA is cleaved forming hairpin structure, hairpin is cleaved by dicer

miRNA processing

pre-miRNA transcribed from DNA which is cleaved to form hairpin structure and cleaved by dicer to form miRNA

miRNA function

binds imperfectly to mRNA inhibiting translation or cleaves it

crRNA production

pre-crRNA is transcribed from CRISPR array > it is then cleaved by CAS proteins and then processed and interacts with the CAS protein to from the effector complex

CRISPR array composition

Palindromes and spacers (foreign DNA)

crRNA function and mode of action:

Described as adaptive RNA defense system. Binds complementary to foreign DNA which signals CAS protein to cleave the DNA

snRNA: location and function

In the nucleus and plays a role in splicing mRNA

snoRNA: location and function

In the nucleosome and mRNA editing, genome imprinting, modifying tRNA and rRNA

Long noncoding RNA

Long RNA molecule that do not encode for proteins but regulate gene expression by binding to proteins, modifying chromatin structure or interacting with mRNA

One gene one enzyme hypothesis

Each gene codes for one unique enzyme

One gene one protein hypothesis

Each gene codes for one unique polypeptide

Codon nucleotide #

3

start codon

AUG

GENETIC CODE RULES

1. Do not write stop


2. include N and C terminus

stop codons

UAA, UAG, UGA

Degenerate

codon coded by more than 1 set of nucleotides

wobble base paring

3rd codon anticodon interaction that is not conventional

Eukaryotic: Core promoter

TATA box in which RNA polymerase will bind as well as accessory proteins

Eukaryotic: Regulatory promoter

Upstream of core promoter and is where other proteins bind

Binding site of ribosomes

A site: Aminoacyl

P site: peptydle site

E site: Exit site

tRNA charging

adding an amino acid to a tRNA

what adds amino acids to tRNA?

aminoacyl-tRNA synthetase

aminoacyl-tRNA synthetase adding check

**Active site**: the active site has specific size and affinity to an amino acid.

**Editing Site**: the editing site will cleave a incorrect aa off

Initiation of Translation in prokaryotes

…

Initiation of Translation in Eukaryotes

\+12 Initiation factor, no N-formal methionine, 5’ cap recognized by small ribosome 40s, no shine-dalgarno sequence but has Kozak instead

Elongation of Translation in prokaryotes

EF-TU-GTP brings charged tRNA to A site, GTP is hydrolyzed and leaves, amino acid on tRNA at P-site transferred to amino acid on tRNA at A-site, a peptide bond is formed by 23s, EF-G-GTP is hydrolyzed moving the ribosome 5-3, then tRNA at P goes to E and the A goes to P.

Termination of Translation in prokaryotes

Stop codon shows up causing release factors bind to A-site which causes hydrolysis of polypeptide and disassembly of 70s ribosome

Draw a replication fork

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Draw the leaf clover model of tRNA