BIO 1100 Final - Mark Grabiner Northeastern University

protein structure and genes

• Proteins are linear chains of amino acids which fold to make complex shapes capable of doing specific task in the cell

• The order of specific amino acids in a protein (primary structure) determines the protein's shape and abilities 

Genes and DNA

DNA holds code for the order of amino acids in a protein 

• Gene: individual unit of genetic information 

• One gene -> one protein (hemoglobin, aquaporin)

• Many traits are influenced by many genes and environment 

• Information in DNA is stored in the order of nitrogenous bases 

• Gene expression: the production of protein based on the sequence of a DNA gene 

what is crucial at every step to making protein?

RNA

Transcription

process that uses the same language but slightly different words

• Changing language from the DNA version to the RNA version. DNA and RNA can base pair with each other so similar comm

translation

RNA sequences are used to figure out how to build amino acids to make polypeptides ot protein. Goes from language of RNA and switches to amino acid language. More complicated than base pairing to make proteins because they do not speak the same language

Building analogy

• DNA is the blueprint for building proteins 

• mRNA is the copy of the blue print you bring to the work site

• Amino acids are the building materials  

• A protein is the finished product

transcription (DNA → RNA) involves

RNA polymerase, template strand, coding strand, promoter, and terminator

RNA polymerase

separates DNA and uses one strand as the template to build on and is essentially the same as the unused coding strand, but has U instead of T

• Adds nucleotides to the 3’ end of the strand they are making. Moves towards 5’ end to add nucleotides to 3’ because it is antiparallel

More capable than DNA polymerase

• Can separate strands on its own - no helicase

• can start its own strand - no primer

promoter

DNA sequence every gene has that allows RNA polymerase to attach to the DNA and begin transcribing RNA

• Nucleotides in a specific order that signal/mark where genes begin and end

• helps polymerase to know which strand is template and which is not

terminators

DNA sequences that mark the end of the gene and stops transcription

translation (RNA → protein) involves

mRNA, terminus, tRNA, Amino-acyl tRNA synthetase, and ribosomes

translation info

RNAs that have codes for making protein sequences are called mRNA and have code for one polypeptide

• mRNA is read in the 5’->3’ direction to build new proteins in the N terminus to C terminus direction
  

Polypeptides has an end terminus with an amino group at one end and a carboxyl group at the other because of the asymmetry of amino acids

5→3, n→ c

translation happens in x to x direction

First amino acid monomer made is at x terminus and last is at the x terminus 

codon

every 3 RNA nucleotides correspond to one amino acids

• Read one after the other, with no overlap

codons and math

Must have 3 nucleotides because there are 20 amino acids used to make proteins and there are 4 base pairs. If you had 3 nucleotides in a row (4^3) you would have 64 coding capacity to code for 20 amino acids which is enough

codon tables

• Left has the letters/nucleotides and right has amino acid they code for

• Start and stop codons mean they have internal signals for start and stop aside from promoter and terminator. These are not the same!!

The first two nucleotides in the codon are most important for amino acids so the third base is the “wobble” base because it is the weakest and it has the least influence on which amino acid is being coded for 

redundant

multiple codons per amino acid

unambiguous

only one amino acid per codon

tRNA

• one end has anticodon to base pair with the mRNA codon and one end has amino acid

• reads codon and supplies amino acid

tRNA function

Reads codon and provides amino acid

amino-acyl tRNA synthetase

Enzyme that synthesizes something

• Specifically recognizes:

  • Amino acid

  • tNRA

• Attaches appropriate amino acid to the tRNA

• ATP is used to power this process of combining amino acids and tRNA

ribosomes

site of translation

• rRNA-protein complex

• Two subunits come together to initiate translation

• Three tRNA binding sites:

  • A site: tRNA amino acid

  • P site: tRNA polypeptide

  • E site: empty tRNA exits

initiation

translation begins at the AUG codon

central dogma

Big idea of molecule biology is that genetic information flows from DNA -> RNA -> protein

• Information in DNA is used to build RNA molecules that contain a copy of that information and then that RNA molecule is read to then build a protein

  • Taking info from one and using it to make the next one 

process of initiation

1. Small subunit of ribosome binds to mRNA

2. AUG codon binds to tRNA to start translation which has the methionine amino acid attached to it

3. Large subunit ribosome comes in and the first initiation tRNA will lodge itself into the p site 

ribosomal recognition and binding in bacteria

mRNA ribosome binding site (shine delgarno sequence) base pairs with rRNA from small subunit, lining up correct AUG start 

ribosomal recognition and binding in eukaryotes

• Small ribosomal subunits binds 5’ cap of mRNA

• Kozak sequence helps ribosome find AUG  5’ RCCAUGG 3’

what helps differentiate start codon (AUG) from methionine?

The secondary sequences that help mark what is the start codon

continuing translation

1. new tRNA with correct anticodon binds to next mRNA codon in A site

2. growing amino acid chain is connected to amino acid from new tRNA

3. old tRNA leaves and message slides over to put tRNA holding peptside in P site and to make room for next tRNA in A site

tRNA gets amino acids from aminoacyl tRNA synthetase

what recruits protein factors that terminate translation?

stop codons

polyribosomes (polysomes)

multiple ribosomes simultaneously translate the same mRNA

how many ribosomes translate the same mRNA?

multiple, simultaneously

eukaryotic gene expression

1. mRNA must be modified before translation

   1. end modificactions

2. eukaryotic mRNA has non coding segments as well as protein coding ones

   1. exons, introns, RNA splicing

end modifications

• 5’ methyl G cap (attached sideways), 3 poly A tail (things that get added to the tain)

• Facilitate export from nucleus, protect from degradation, facilitate translation (ribosome recruitment)

exons

protein coding (expressed; exit the nucleus)

introns

non protein coding (intervening)

RNA splicing

remove of introns, connect exons

• Takes place in nucleus where introns are removed and exons are combined 

• Spliceosome

spliceosome

protein, RNA complex that carries out RNA splicing 

snRNPs

• Small nuclear ribonucleoproteins

• snRNA (small nuclear RNA) + protein 

  • Base pairs with sections of the introns

where are ribosomes found

• Cytoplasm - cytoplasmic proteins 

• Attached to ER - proteins found in membranes, certain organelles, and secreted outside of cell

how do ribosomes know where to bring membrane proteins, etc to the rough ER?

signal peptides

where does translation always begin and what can redirect it?

starts in cytoplasm but peptide sequence can redirect to the ER

signal peptides

amino acid sequence that stalls translation and targets ribosomes to ER

• Recognized by the signal recognition particle (SRP)

• Escorts to ER bound receptor, translation resumes

First amino acids made from end terminus of the protein is the protein recognized by the SRP and will bind to any polypeptide with that signal peptide and bring the whole thing to channels on the outside of the ER and the ribosome will dock wit that channel and make the proteins in the lumen of it and then it will enter a vesicle and can go to the golgi or somewhere else at this point 

Removed after it did its job

what two factors allow prokaryotic genes to be transcribed and translated at the same time?

they do not have intros or a nucleus + the order of how genes are made and translated

mutations

changes in DNA sequence of a gene

• substitution, deletion, insertion

substitution

one nucleotide is replaced by another 

• missense and nonsense

deletion

one or more nucleotides are removed from the gene

insertion

one or more nucleotides are added to the gene

what does “missense” mean

wrong amino acid, sometimes impactful but sometimes neutral

what are silent mutations?

errors with substitution where you have the same amino acid even though you have a different codon

nonsense

stop codon is created

frameshift mutations

these deletion/insertion mutations cause more drastic changes than substitution. They cause a different set of codons to be read after the mutation (a different reading frame)

some sources of DNA mutations

• DNA replication

• Mutagens: substances that cause DNA mutations (UV light and chemical mutagens)

what is the importance of variety in proteins for cells types?

the different active proteins present in a cell determine the type of cell and what it can do

what allows for the production of different proteins?

utilization of different genes

what system affects the amount of active proteins are in every cell?

tight regulation during each step from gene to protein

what step of gene replication is the most common way to control a protein’s presence?

gene transcription

transcription factors

bind to very specific DNA sequences near the genes they control - must know where the gene is

• increase gene expression by bringing RNA polymerase to a gene’s promoter

• decrease expression by preventing RNA polymerase from attaching 

what marks the start of gene expression with transcription?

promoters

what allows for TF to know where to bind to DNA / know where the gene is

regulatory DNA sequences

because TF have different genes expressed in proteins, they have different ____

active transcription factors

constitutive expression

gene expressed 24/7

inducible expression

gene is off until actively turned on

• positive control allows for transcription to start

repressible expression

gene is on until actively turned off

• negative control stops transcription

operons

multiple genes with one promoter, only found in prokaryotes

lac operons

contains genes needed for lactose metabolism under a single regulated promoter - only one mRNA is used to make this protein and has multiple start and stop codons 

Catabolite activating protein exerts _____ control

positive - activates expression from operon

lac repressor transcription factor exerts ___ control

negative - stops gene expression

the repressor is negatively regulated when lactose is ____

absent

what happens with lac repressor transcription factor when there ISN’T any lactose

binds to the operator and that prevents the polymerase from attaching to the promoter

what happens with lac repressor transcription factor when there IS lactose

then the repressor does not bind to the operator which allows polymerase to bind to the promoter

Positive regulation of the lac operon by CAP occurs when

glucose levels are low

what happens with CAP when there IS cAMP

CAP binds to promoter and increases RNA polymerase activity 

what happens with CAP when there ISN’T any cAMP

CAP does not bind to promoter - transcription occurs at low rate 

presence of glucose means what for cAMP and CAP

no cAMP and CAP is not activiated

what happens to CAP when glucose is high?

CAP is low

what happens to CAP when glucose is low?

CAP is turned on

Strong expression of the lac operon requires____

release of repression AND transcriptional activation 

mutations to regulatory DNA sequences can cause genetic disease and cancer by changing the availability of _____

certain proteins in different cells

why would a cell need to destroy a protein?

• Remove misfolded proteins

• Remove proteins designed to their job for only a short period of time

• Destroy proteins needed for cell to change behavior

mating switches require changes in____

gene expression and protein removal 

Ubiquitin protein chains mark proteins for what?

for destruction

• also used to target cyclins for degradation

what is a proteasome?

an enzymatic protein degradation complex targeting ubiquitylated proteins 

what are peptide fragments from proteasomes used for?

they are displayed to immune cells to detect intracellular pathogens 

methods of manipulating DNA

polymerase chain reaction, DNA sequencing, restriction enzymes and CRISPR system enzymes

polymerase chain reaction

Method for making DNA sequences using DNA polymerase in a test tube 

• Molecular cloning, diagnostic, forensics, DNA sequencing, gene expression analysis, etc

DNA sequencing

• Tells order of nucleotides in a DNA samples

• Helps to discover genes for a diagnosis

Restriction enzymes and CRISPR system enzymes

• Cut DNA at specific locations

• Helps make genetic changes and for diagnostics

problems with PCR tests

same as within the cell, there are limitations of DNA polymerase. main issues are:

1. cannot unwind double stranded DNA

2. cannot start chain, only add to existing strand

what is the PCR primers job?

to initiate replication

aspects of PCR primers

• Short DNA sequence (oligonucleotides) produced synthetically and added to PRC reaction

• Do not need primase

• Get to define which section gets copied (the amplicon) 

• PCR primers base pair with sections on both strands

direction of PCR primers

Extend towards direction of other primers to they are a template in the next round - have to go in direction so 3’ end gets extended into area you want replicated 

PCR components

• Template (source) DNA

• Oligonucleotide primers

• DNA polymerase

• DNA nucleotides (dNTPs)

• buffers/salts 

PCR cycle

1. Denature - 95C

2. Anneal (prime) - 50-60C

3. Extend - 72C

4. Repeat 

• Each new strand you build is a template for the next strand you build

denature - pcr cycle

Split DNA strands (break H bonds with heat)

anneal (prime) - pcr cycle

1. Reduce heat, sequence specific primers bind (anneal) to target DNA

   1. Primers define amplification religion (“amplicon”)

extend - pcr cycle

DNA polymerase extends from primer using target at template 

repeat - PCR cycle

go through denature, anneal, extend again

• Each new strand you build is a template for the next strand you build

Heat stable polymerase

Taq is a bacteria with heat stable polymerase (does not denature during heating of DNA to separate strands) which is why PCR works

DNA cloning uses____

bacteria to propagate pieces of DNA