genetics unit 3 + cumulative

the _______ making up DNA contains the information for making proteins

linear sequence of deoxyribonucleotides

mRNA from DNA

• one of the two strands of dna serves as a template for the information to be transcribed into mRNA

• mRNAs associate with ribosomes to ultimately produce proteins

the genetic code is written in linear form using the ribonucleotide bases that compose mRNA molecules as ____

“letters”

the sequence of RNA is derived from

the complementary bases in the DNA

in mRNA triplet codons each specify

one amino acids

the code in mRNA contains what kind of signals?

• stop and start

• certain codons are necessary to initiate and to terminate translation

why is the code triplet for 20 amino acids?

4³ = 64 which is just enough with a few extra

4² would be too little and 4^4 would be too many

the genetic code is

• non overlapping

• comma-less

• degenerate

• unambiguous

• nearly universal

unambiguous vs degenerate for the genetic code

• unambiguous = each triplet specifies only a single amino acid

• degenerate = more than one triplet can code for the same amino acid

what infers that the genetic code is non overlapping and comma less

• the fact that the code reads three nucleotides at a time in a continuous, linear manner

this represents

frameshift mutations

RNA homopolymers are

RNA nucleotides with only one type of nucleotides

who discovered / how did they discover which amino acids were encoded by specific codons

• Nirenberg and Matthaei

• added homopolymers to an in vitro translation system

• poly U directed the incorporation of only phenylalanine - they discovered that UUU codes for phenylalanine

poly a and poly c

poly A= lysine

polt c= proline

the genetic code is degenerate, what does this mean?

• many amino acids specified by more than one code

• 64 different triplet codes but only 20 amino acids

what are the only two amino acids that are encoded by a single codon

• tryptophan and methionine

• AUG - met

• UGG- trp

the wobble hypothesis

• proposed by Francis crick

• predicts that hydrogen bonding between codon and anticodon at the third position is subject to modified base pairing rules

what three codons serve as termination codons and do not code for any amino acid?

UAG, UAA and UGA

what are some exceptions to the universal genetic code?

• mitochondrial DNA

mitochondrial DNA can express UGA as

tryptophan instead of termination

CUA can be altered in mitochondrial dna to be read as

threonine instead of leucine

AUA can be read as what in mitochondrial DNA

methionine instead of isoleucine

AGA can be read as what in mitochondrial dna

termination instead of arginine

AGG can be read as what in mitochondrial dna

termination instead of arginine

rna synthesis overview

• no primer is required for initiation

• the enzyme uses ribonucleotides instead of deoxyribonucleotides

• transcriptions begins with template binding by RNA polymerase at a promoter

• the o subunit is responsible for promoter recognition

prokaryotic transcription begins at the transcription start site where

the dna double helix is unwound to make the template strand accessible

e.coli promoters that have two consensus sequences _______ and _____ positioned at -35 bp and -10 bp with respect to the transcription initiation site

TTGACA TATAAT

in Ecoli once initiation has been completed with the synthesis of the first 8-9 nucleotides ….

sigma dissociates and elongation proceeds with the core enzyme

at the end of the gene in e coli, transcription terminates when a termination sequence is reached at ~40 bp and

transcribed into rna, forming a hairpin structure (self complementary)

transcription in eukaryotes

• occurs in the nucleus and not coupled to translation

• requires chromatin remodeling

• promotors, enhancers also influence transcription regulation

what is required of eukaryotic mRNAs to produce mature mRNAs?

processing

what is the shortened version of RNA polymerase II?

RNP II

difference between start codons in prokaryotes and eukaryotes

both use AUG

• prokaryotes it represents n-formylmethionine (feet)

• eukaryotes, unformulated methionine is used

what are the three termination codes and what do they stand for

• UAG, UAA. UGA

• they do not code for any amino acids

What are the key components of the RNA Polymerase holoenzyme in prokaryotes?

• core enzyme: responsible for elongation

• sigma subunit: responsible for promoter recogninition and template binding

• no primer is required for rna synthesis

identify the two consensus sequences in E. coli promoters

-35 region:TTGACA

-10 region: TATAAT (pribnow box)

describe the mechanism of prokaryotic termination

• it occurs when a termination sequence (approximately 40 bp) is reached

• the transcribed RNA forms a hairpin structure due to self-complementarity which causes the polymerase to dissociate

how does the location of transcription differ in prokaryotes from eukaryotes?

in prokaryotes: translation and transcription are coupled in the cytoplasm

how does eukaryotic transcription differ from prokaryotic?

• eukaryotic occurs in the nucleus

• requires chromatin remodeling to access dna

• involves regulation with both promoters and enhancers

• produces an initial transcript hnRNA that requires extensive processing

what does the wobble hypothesis suggest?

• the first two positions of the codon require strict base pairing but the third position of the anticodon interaction is subject to modified base-pairing rules, allowing one tRNA to recognize multiple codons

what is the initial transcript for eukaryotes that requires extensive processing

• hnRNA (heterogeneous nuclear RNA)

describe the components of a eukaryotic core promoter?

TATA Box: located at -35 bp (TATAAAA)

• facilitates DNA denaturation to determine the start side

CAAT Box: located -80 bp

• influences the efficiency/ rate of transcription

What are enhancers and where are they located?

• they are cis-acting elements that modulate transcription from a distance

• found upstream, within introns or downstream of gene

distinguish between general and specific transcription factors

• general factors: absolutely required for RNP II transcription (TFIIA, TFIIB, TFIID)

• Specific factors: influence the rate and efficiency of transcription for specific genes or groups of genes

what transcription factor specifically binds to the TATA sequence?

TFIID

CAAT box

• promoter element

• -80 upstream

• helps efficiency

Describe trans acting factors that facilitate RNP II binding

• usually a protein coded for by another gene

• gen transcription factors are required for transcription by RNP II

• specific TFs influence rate and efficiency of transcription and may be specific to gene or group of genes

in humans what are the transcription factors?

TFIIA, TFIIB, TFIID

which transcription factor binds directly to the TATA sequence?

TFIID

cis- promoter elements that interact with RNP II - how does it begin transcription?

RNP II recognizes a core primer element in the DNA sequence that will determine where RNP II binds and begins transcription

TATA box in eukaryotes

• core promoter element located at -35 bp upstream from the start of transcription

• consensus sequence TATAAAA

• helps facilitate denaturalization of helix so helps determine transcription site

in transcription in eukaryotes the first rna produced is

hnRNA (heterogeneous nuclear RNA)

• also called primary transcriptt

what does RNA polymerase ii do?

makes mRNA

Core promoter=

the exact spot where transcription begins

introns in rna are removed by

splicing

in rna processing what does the 7-methylguanosine cap do?

• protects the 5’ end of mRNA

• transports mRNA out of the nucleus

what does the poly-a tail do in rna processing

• has up to 250 residues

• protects RNA from degradation

introns and rna

• removed from rna

• they are not expressed in the amino acid sequence of the protein

give overview of RNA processing

dna template → (transcription)→ pre mRNA→ (cap and tail added)→ processed pre-mRNA → spliced mRNA

exons and introns in mature mRNA

introns removed, exons joined together

• therefor, mRNA is usually smaller than initial rna

translation

• the biological polymerization of amino acids into polypeptide chains

translation requires

• amino acids

• mRNA

• ribosomes

• tRNA

ribosomes

• large, intricate and numerous cellular structures

• each has a large sub unit and a small subunit

eukaryotic vs bacterial cells and ribosomes

• one bacterial contains about 10,000

• eukaryotes have many times more

what are ribosomes composed of

• ribosomal proteins and rRNAs

____ provide for important catalytic functions associated with translation

rRNAs

what do ribosomal proteins do?

• promote binding of various molecules involved in translation?

rRNA genes are also called

ribosomal DNA

rRNA genes are ______ repeated

moderately repetitive and tandemly

ecoli genome and rRNA

• has 7 copies of a single sequence that encodes all 3 rRNA components

what are the three rRNA components for prokaryotes?

16S, 23S, 5S

Eukaryotes and rDNA

• MANY more copies than prokaryotes

• present in clusters at various chromosomal sites

a single transcript of rDNA encodes for

three of the components

28S, 18S, 5.8S

In eukaryotes there is a unique component of rRNA that is encoded by a distinct gene cluster and located separately - not part of the larger transcript. what is it?

5S rRNA

trna function can be described as

• adapter molecules to adapt the triplet codons in mRNA to the correct amino acid

tRNAs overview

• composed of only 75-90 nucleotides

• identical structure in bacteria and eukaryotes

• transcribed as larger precursors but cleaved into mature 4S molcules

• several nucleotides are modified through post transcriptional modification

what activates tRNAs with the appropriate amino acid?

Aminoacyl tRNA synthetase

how many different enzymes are each responsible for linking a different amino acid to its corresponding tRNA molecule?

20!

what are the three steps of translation?

• initiation

• elongation

• termination

what does initiation require?

• small and large ribosomal subunits

• GTP

• charged initiator tRNA

• mg2+

• initiation factors

initiation of translation steps

• 1. mrna and IF1 bind to small subunit

• 2. IF2 and IF3 bind to complex while initiated tRNA binds to mRNA codon in the P site

• 3. large subunit binds to complex, IF factors are released EF-Tu binds to tRNA, facilitating entry into A site

what does elongation require in translation?

both ribosomal subunits assembled with the mRNA to form the peptide site and aminoacyl cite

elongation in translation steps

1. trna enters A site

2. peptide bond forms

3. ribosomal shifts (translocation)

what happens when tRNA enters the a site in elongation

a charged tRNA with an amino acid matches its anticodon to the mRNA codon

what happens after the trna enters the A site in elongation of translation

• the amino acid in the Psite is transferred to the amino acid in the A site, growing the chain

describe translocation in elongation

• ribosome moves 1 codon forward via gTP

• trna moves A site to P site and p site to e site

describe all the sites of elongation in trna elongation

a site= arrives p site= peptide bond e site= exit

termination is signaled by what in the A site for translation

• stop codons

what are the stop codons

• UAG, UAA, UGA

what helps cleave the polypeptide chain from the trna to release it from the translation complex?

GTP dependent release factors

eukaryotes and translation

• ribosomes are larger than in bacteria

• transcription and translation are spatially and temporally separated

• requires more factors for initiation, elongation, termination than bacteria

• ribosomes are associated with the endoplasmic reticulum

in prokaryotes ribosomes are

free floating

proteins and diseases through translation

• accumulation of intermediates due to metabolic deficiencies (Archibald Edward Garrod)

• inborn errors of metabolism

• incest related

beadle and Tatum

• showed rear nutritional mutations in the bread mold neurospora caused the loss of enzymatic activity that catalyzed an essential reaction in wild type organisms

neurospora

• a fungus

• in the wild type like bacteria can be grown on minimal media

• can be used to study genetic mutations of nutritional mutants

• mutations can be generated at high frequency using radiation

studies of neurpspora led us to the one gene on enzyme hypothesis, describe it

• not all proteins are enzymes and some proteins have more than one subunit

• this led to us actually ending up to one-gene:one polypeptide chain

how can you tell if a neospora has a mutation?

• does not grow on minimal medium

• grows on complete medium

neospora can tell us what genetic mutation has occurred by

• adding each nutritional value to a minimal media until it grows

• shows which exact mutation occurred

spontaneous mutations

• happen naturally and randomly

• are usually linked to normal biological or chemical processes in the organism