Unit 3

Chapter 8

Intro

DNA = instructions for how to synthesize proteins
Reading a DNA template to make an RNA copy (transcription) DNA nucleotides to RNA nucleotides
Decoding the RNA to make a protein (translation)
Once RNA copy is made, it has to be decoded, nucleic acid → amino acids
Synthesis and anabolic pathways

Transcription components; RNA polymerase

Most important component is the enzyme that catalyzes the reaction
RNA polymerase: has different parts
When all the parts are together it is called the holoenzyme
The holoenzyme is made up of core polymerase and sigma factor
Core polymerase: made up of different subunits, 2 alpha, beta and beta prime and an omega part form the core
Each subunit has its own function
2 Alpha: bringing all the subunits together and holding them in place
Beta and beta prime: sits on top of alpha and forms a channel where the DNA template will sit in the polymerase in between the beta and beta prime
Catalytic site: sits in the beta prime subunit, when binding nucleotides together it takes place here
Sigma factor: can attach and detach to the core polymerase, its function is to find the promoter region
Promoter: sequence of nucleotides on the DNA that is right in front of the start of the gene, polymerase has to find this spot to start coding so sigma factor finds the promoter of the gene

RNA polymerase

Sigma factor: in bacteria, there are tons of sigma factors, different types of them
Every sigma factor is usually responsible for identifying a specific promoter
In E. coli, a sigma factor is called sigma-70
Sigma-70 identifies a family of genes called the housekeeping genes
Housekeeping genes: always there and being transcribed because the protein product is needed to perform day to day functions
Each housekeeping gene has a promoter that sigma-70 is recognizing
If there is a different family of genes a different sigma factor is used
Promoters can be made up of big sequences of nucleotides, what specifically is sigma-70 or sigma factors binding to?
Sigma factors only see a small part of the promoter, they recognize the consensus sequences
Consensus sequences: the part of the promoter that the sigma factor is recognizing, the consensus sequences for sigma-70 are position -10 and -35
Positions: if the start of the gene is the part closest to the sequence (let's say gene A), that is plus 1, then moving away from the start of the gene is negative, 10 away is -10 position
Looking for something, not identical, but something similar to the consensus region
Small changes in consensus sequences can affect transcription
Changing the sequence can down regulate transcription meaning transcription efficiency would be less
Up regulate transcription: can benefit the efficiency of transcription based on the mutation occurring
As the sigma factor is scanning, when it finds the right positions, the structure of DNA and shape affects the reading of consensus sequence recognized

Defining a Gene

Upstream: gene A is upstream of gene B, promoter is upstream of gene A
Downstream: gene B is downstream of gene A, terminator region is downstream of gene A and gene B
+1 is upstream of the gene, gene does not start at +1 but it is transcribed
mRNA: single stranded
+1 region is transcribed and is important in regulating the translation step
+1 region is untranslated and is called the leader and contains ribosome binding site for translating gene A transcription
You can have multiple genes transcribed on the same RNA molecule for bacterial cells
Gene expression: this means it is being expressed as a protein, the cell has done transcription and translation and we have the protein product

Transcription of DNA to RNA

Transcription occurs in three phases

Initiation
Elongation
Termination

Initiation

Holoenzyme is present
Double stranded DNA with promoter and consensus sequences
Core polymerase and the sigma factor
Holoenzyme scans DNA and uses sigma factor to find and identify the promoter
Holoenzyme is loosely bound to the DNA
Once sigma factor finds the promoter it will bind tighter to the DNA
Closed complex: scanning is over, holoenzyme binds tighter
Polymerase will start unzipping the DNA, as it unzips the nucleotide bases become exposed
Will read the first couple of nucleotides and bring in the complement
First ten to fifteen are read and synthesized as mRNA, at this point the sigma factor leaves
All that is left is the core polymerase
Initiation is completed when sigma factor leaves

Elongation

Takes place within the transcription bubble
Polymerase has unzipped a portion of the DNA, exposing the nucleotide bases
Polymerase will continue to read the nucleotides and bring in the complement ribonucleotides
5’ -> 3’ direction
Sequential addition of ribonucleotides from nucleoside triphosphates performed based on complementary base pairing
Synthesis reaction, synthesizing mRNA, the bond formed is a phosphodiester bond
Every time a ribonucleotide is brought in it forms a phosphodiester bond with the next one
Topoisomerase: uncoil the circular, folded DNA ahead of the polymerase so the polymerase can move smoothly

Termination

There are two types of transcription termination
Rho-independent and Rho-dependent
Rho: an enzyme that is in a family called the helicases, helicases help unzip nucleotides, can break hydrogen bonds holding nucleotides together
Rho dependent: polymerase pauses and allows Rho (enzyme helicase) to hop onto the DNA and glide/slide along the mRNA until it gets to the polymerase which destabilizes it and falls off the DNA. Rho will then use its helicase activity
Hydrogen bond between nucleotides of DNA and ribonucleotides of mRNA (DNA/RNA hybrid), so we need the Rho to break those bonds apart and remove the messenger RNA
Rho independent: genes that don’t use this enzyme, based on sequences in the gene that is transcribed onto the mRNA, two important ones G-C rich loop and poly u site

G-C rich region: codes for the region on the mRNA it causes the mRNA to make a secondary structure known as a loop, loop recruits other proteins to destabilize polymerase and knock it off the dna
Poly u site: a bunch of A’s code for a bunch of U’s that means its easy for the cell to unzip. U and A have two hydrogen bonds so it requires less energy to unzip, need poly-u site to ensure that the RNA/DNA hybrid is unzipped

Rho independent needs these two regions

Antibiotics that affect transcription

Rifamycin B: selectively binds to the bacteria RNA polymerase of bacteria and inhibits transcription initiation and will not target eukaryotic cells
Actinomycin D: nonselectively binds to DNA and inhibits elongation, inhibits polymerase from unzipping the DNA, bacterial DNA and human DNA are very similar so actinomycin was inhibiting growth of human cells… bad
Use it for a cancer drug to inhibit tumor cell growth

Transcription in Archaea and Eukaryotes

Across all three domains transcription of DNA into RNA is very similar
Multisubunit DNA dependent RNA polymerase is used in all transcription, holoenzyme and alphas and betas, sigma factor etc,
Archaea and Eukaryotes differ from bacteria in the termination and initiation stages
Archaea and eukarya dont have a sigma factor
The TATA binding protein recognizes motifs in the promoter called the TATA box
The initiator proteins remain at the promoter or are removed before elongation begins
Archaea and bacteria utilize operons
Operon: a group of two or more genes transcribed at the same time from the same mRNA molecule
Eukaryotic cells have different polymerases for transcribing different RNA, bacteria use the same polymerase for all RNA types

Six classes of RNA

Messenger RNA: instructions to make a protein
Ribosomal RNA: makes up the ribosome
Transfer RNA: also used in protein synthesis to bring in amino acids in the protein
Small RNA: important for regulating transcription and translation
tmRNA: help unstick damaged ribosomes, mRNA can get stuck in a ribosome so tmRNA can come in and unstick it and release mRNA
Catalytic RNA: have enzymatic function, can perform a chemical reaction in a cell
RNA is unstable and can die within minutes

The genetic code

Consists of nucleotide triplets called codons
There are 64 possible codons
DNA to RNA brings in complements
RNA to protein, this requires nucleotides going to amino acids and those are not complements
61 specify amino acids: sense, includes the start codons
3 are stop codons: nonsense codons
Only 4 nucleotides, uacg, and you need them in group of 3, 4³ = 64
The codons tell us what amino acid will be
20 amino acids (alanine, arginine, asparagine, glycine, lysine, phenyalanine, glutamate, cystine, histine, proline, serine, tryptophan, valine, tyrosine, threonine, leusine, glutamine, methionine, ileusine,

Organization of the code

Code degeneracy: multiple codons that can encode for the same amino acid, more codons than amino acids
The code operates universally across species with very few exceptions, some yeast species with a handful of amino acids, and our mitochondrial DNA
aug is the start codon and codes for methionine
UAG, UAA, UGA are stop codons

Translation

Synthesis of protein or polypeptide
It is directed by the sequence of nucleotides found in the mRNA
Triplet codons that code for the amino acids that will be used in the synthesis of the protein
There is directionality in translation
N terminal → C terminal
Amino acid has an amino group, carboxylic acid group and a central carbon, and an R group that differentiates it
At the beginning of every protein there is an exposed amino group and at the end

3 types of RNA used in translation

Directly functioning in translation
mRNA, rRNA and tRNA
mRNA: the transcript, copy of DNA and now we have mRNA, holds the triplet codons
tRNA: brings amino acid to site of protein synthesis, catalyzed by aminoacyl-tRNA synthetases, has secondary structure, not linear, contains the anticodon (complement to a codon on mRNA) and amino acid at the top of the tRNA, if the codon codes for methionine, the amino acid carried on tRNA should be methionine
Aminoacyl-tRNA: places the correct amino acid on the correct tRNA
rRNA: contains a small 30S subunit, a larger 50S subunit that come together and form 70S ribosome, also contains protein subunits, there are 20 proteins in the small subunit
16s rRNA: on small subunit
Large subunit: has 23S rRNA and a 5S rRNA
23S rRNA: catalytic site on the ribosome for protein synthesis, rRNA enzyme ribozyme

The 70S ribosome

Binding sites for tRNA (three)
A site: acceptor site, all new tRNAs with an amino acid come in, also referred to as the amino-acyl site
P site: peptidyl tRNA site, has the tRNA that has the growing polypeptide chain attached to it
E site: exit site, where the empty tRNA (no longer has an amino acid) will leave through this site when it drops off amino acid

Translation

Takes place in three steps
Initiation, elongation and termination (same as transcription)

Initiation of translation

Protein synthesis
Ribosome subunits will be here
Other proteins that help this process (Initiation factors (IFs))
mRNA
Initiator tRNA: always the first tRNA that comes into translation
Different from other tRNAs because it always has methionine on it (start amino acid) and a formal group on the methionine/amino acid, fMet (formal methionine) tRNA, unique to bacterial domain
Archaea and eukaroytes do not add a formal group

Process of initiation

mRNA has to be present
Small subunit of the ribosome (30S) will bind to the mRNA
Very specific how it binds and where it binds and it is helped by the initiation factor 3
IF₃: guides the small subunit to the correct spot to the mRNA near the start codon
Upstream of the start codon is the shine-dalgarno sequence where the IF₃ will help the ribosome bind
When the ribosome is seated it has the start codon in the middle called the p site
Initiation factor 1 comes in and binds to the mRNA, the next triplet codon downstream of the start
Blocks tRNA from coming into that codon
tRNA carries formal methionine and is guided to the mRNA and small subunit by the initiation factor 2, have amino acid, and anticodon which is complementary of the codon on the p site
Binds to the complement base pair
This is the only tRNA that binds to the ribosome on the p site
IF₃ leaves
Large subunit of ribsome comes in and binds to the small subunit and now all the sites are formed, E, P and A
Requires energy to bring large subunit and lock everything in place: GTP → GDP to release the other initiaton factors
Once initiator tRNA is in place, initiation is done

Elongation

Cycle of events
Adding each amino acid to the growing polypeptide chain done in a specific sequence
Based on codons on mRNA that correspond to a specific amino acid
Three steps that repeat over and over again

Aminoacyl-tRNA binding: when the next amino acid comes into the ribosome
Transpeptidation reaction: where the peptide bond is formed between the two amino acids
Translocation: when the ribosome moves down the mRNA by one codon and then step 1 starts again

This cycle happens for every single codon
Just like initiation there are proteins
Elongation factors (EFs)
Also involves all three ribosomal sites, p e and a

Step 1: bringing in the next aminoacyl tRNA with amino acid and it binds to the a site, it is coding for on mRNA and the anticodon matches c-g, a-u, EFTU helps with this and guides in the aminoacyl tRNA to the A site

Step 2: transpeptidation reaction, amino acid 1 in p site and acid 2 in a site bind together forming a peptide bond, catalytic reaction being catalyzed by the ribosome, the 23SrRNA peptidyltransferase

Step 3: translocation, moving the ribosome down one codon so A site goes to P site and P site goes to E site and the A site is now on the new codon, amino acid chain is now on the tRNA from the A site and the one on the p site is now empty. EFG helps with the movement

Translocation has three steps

Peptidyl tRNA with the growing polypeptide chain is now p site as ribosome moves
Empty tRNA without amino acid is no in the e site and leaves
Empty A site to accept a tRNA

Termination of protein synthesis

Takes place at any one of three codons
When the ribosome translocates it comes up a stop codon in the A site
Nonsense codons: UGA, UAG, UAA, NO TRNA
Release factors (RF): aid in recognition of stop codon to release ribosome, mRNA and protein, in prokaryotes there are three RFs, eukaryotes only have 1
Release factor 1 or 2 enters the A site and the polypeptide chain is released
Release factor 3 binds to the and A site and rf1 leaves
Elongation factor (EFG) comes in with RRF (ribosome recycling factor) and kicks out the RF₃ and unlocks the two ribosomal subunits (small and large are separated) then the mRNA is released
IF3 binds to the 30s subunit to start over with initiation
Protein gone, large and small units released, mRNA freed NOTHING IS DEGRADED

Antibiotics that affect translation

Can target just bacterial cells
Erthyromycin: doesnt allow ribosome to move down the mRNA
Tetracycline: inhibits aminoacyl tRNA to A site (where 16s rRNA is)
Chloramphenicol: inhibits peptide bond formation which is inhibiting peptidyl transferase activity in the 23s rRNA site

End of translation

We have a protein
Protein can be a structural protein, an enzyme or anything the cell needs
Protein needs to get folded into the correct structure

Protein folding

Chaperones: takes newly synthesized protein and makes sure it folds correctly
GroEL and GroES: form stacked rings with a hollow center, as linear protein is synthesized these rings are stacked on it so it doesn’t fold before the whole protein is made
DnaK: do not form rings, clamp down on the polypeptide to assist folding and make sure the linear protein does not fold on itself too early