Protein Synthesis

I. Protein Production A. Background Info

2. Ribosomes (in the cytoplasm) are where ALL proteins are initially produced:

- proteins staying inside cell will be completed here

- proteins to be exported or that become lysosomes are transferred to ribosomes on RER and synthesis is finished there

1. DNA (in the nucleus) is the blueprint for creating proteins

3. RNA carries out P.S. by acting as a messenger b/n DNA & ribosomes.

B. RNA v. DNA

1. RNA is single-stranded 2. RNA is found in the nucleus & the cytoplasm 3. RNA is also a nucleic acid made up of nucleotides consisting of 3 parts: a. phosphate group b. pentose sugar c. nitrogenous base

HOWEVER…

Ribose DEOXYribose

new base pairing rule:

A = U C = G

4. Three main kinds of RNA:

a. mRNA – carries info from DNA to ribosomes b. tRNA – carries amino acid from cytoplasm to ribosome c. rRNA – help build ribosomes; binds mRNA and tRNA together to make polypeptide chain

C. CENTRAL DOGMA

D. Protein Synthesis 1. Transcription - Takes place in the Nucleus WHY?

Taking info found in the DNA and turning it into a molecule of mRNA

As in replication, the DNA must unwind; however, ONLY ONE strand of DNA is used as a template, the other remains untranscribed

*** DNA IS TRANSCRIBED 3’ to 5’

RNA IS SYNTHESIZED 5’ to 3’

a.Initiation 1. RNA Polymerase – the enzyme that binds to DNA and transcribes it into mRNA.

~20 base pairs of DNA are unwound and then RNA poly reaches start site and begins transciption

2. Promoter - a specific sequence of nucleotides on the DNA that tells RNA poly “bind here” this sequence is known as the “TATA box” (~25 bases upstream from the gene to be transcribed) RNA polymerase will orient itself here

b. Elongation RNA Polymerase moves along and “reads” the DNA adding complimentary RNA nucleotides (Chargoff’s base-pairing rules)

c. Termination (Fig. 1) RNA Poly continues until it reaches a “stop signal” sequence RNA Poly will detach and release the new mRNA This new mRNA (i.e. transcript) now carries the instructions for making proteins

mRNA leaves the nucleus and goes where?To a ribosome in the cytoplasm

IMPORTANT: Before the new mRNA moves to the cytoplasm, SPLICING occurs

1. INTRONS:

2. EXONS:

segments of DNA that are cut out b/c they do not code for any part of a protein

segments of DNA that are EXpressed code for proteins

Non-coding regions of DNA

Coding regions of DNA

mRNA Processing (i.e splicing)

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html

Promoter region

2. Translation

translating the info on mRNA into amino acids (i.e. polypeptide chain protein)

mRNA is translated 5’ to 3’

based on the Genetic Code…

CODON - a series of 3 mRNA nucleotides that specify: 1. a particular amino acid

2. a “start signal” (only one) 3. a “stop signal” (3 different)

(64 possible codons)

Example:

If a gene is 99 DNA base pairs long ~~~~~~~~~ (99) The mRNA is also 99 base pairs long ------------------ (99)

The protein will have 33 amino acids @@@@@@ (33)

99/3 = 33

Other things needed for translation:

1. Ribosomes: contain two subunits, large (heavy) and small (light)

Interesting fact:

- Ribosomes have 3 sections you APE:

A = Acceptor site: where tRNA enters P = Peptidyl site: where one amino acid is bonded to another in the polypeptide chain E = Exit site: where tRNA molecule leaves after dropping off its amino acid

2. tRNA’s: carry the amino acids - they contain a region called the ANTICODON: a sequence of 3 nucleotides that is complimentary to the codon on the mRNA - where tRNA binds to mRNA

a.Initiation Ribosome subunits recognize and bind to a recognition sequence on mRNA Ribosome then begins moving along mRNA in a 5’ to 3’ direction - translation initiates when ribosome reaches the “start” codon (AUG) the first amino acid (METHIONINE) enters the P site, the ONLY amino acid to do that.

b. Elongation The ribosome moves along the mRNA and new amino acids (carried by tRNA) are added forming a polypeptide chain Amino acids are linked by peptide bonds

c. Termination (Fig. 2) The ribosome reaches one of three “stop” codons (i.e. there is no complimentary tRNA anticodon) No more amino acids can be added so the ribosome detaches & releases new protein

Signal sequence will determine what proteins are finished being synthesized on the ribosomes ofthe RER

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html

II. Mutations – Changes in the nucleotide sequence of DNA. Two general categories:

A. Point Mutations: mutations of single genes 1. base-substitutions (one base for another) two kinds: a. transition: purine for purine (AG)

pyrimidine for pyrimidine (CT) b. transversion: pyrimidine for purine (CA) or vice versa

2. Frameshift Mutations involve the insertion or deletion of one or more nucleotides from DNA causes a shift in the reading frame almost always lead to non-fxning protein

e.g. THE CAT ATE THE RAT

deletion of C

THE ATA TET HER AT

base-substitutions & frameshifts are either: 1. silent – code for the same amino acid

2. missense – code for a different amino acid

3. nonsense – code for a stop codon (can lead to nonfxning protein)

Silent mutation

Missense mutation

Nonsense mutation

B. Chromosomal Mutations

chromosomes may break during replication and rejoin in abnormal ways

4 specific types (examine during genetics)

Remember:

Changes to the amino acid sequence probably changes the three-dimensional shape of the protein. Since protein function if highly dependent on shape, this will lead to impaired fxn or possibly complete nonfxn of the protein!

If an individual inherits mutated genes for a single protein, and if that protein is essential for life, the individual may have seriously impaired health or may even die

Examples: hemophilia, sickle-cell anemia, cystic fibrosis, Huntington’s

III. Gene Expression A. Gene Regulation, WHY?

--Why don’t organisms just express every gene in their genome all the time?

--Bacteria (E. coli), for example, live in a wide range of env’tal conditions and it is more efficient to express only those genes that are necessary for survival

--Remember, a high amount of NRG is involved in gene expression (i.e. TXN & TLN)

--THUS, genes need to be regulated

B. How are genes regulated? (Prokaryotic Mechanisms)

1. Genes responsible for a given cellular fxn are organized into operons

2. These operons may be turned on (inducible) or turned off (repressible) depending on the situation

3. EXAMPLE: Lactose Metabolism (Inducible) Tryptophan sythesis (repressible)

Operator – Promoter –

Repressor –

Structural Genes -

DNA

The Lac Operon: An Inducible System

where RNA Polymerase binds to DNA to begin transcription (1 per set of genes)Protein that blocks RNA Polymerase from binding, thus NO txn & NO gene expression(comes from repressor gene)

the On/Off switch of a particular gene

Indicate the primary structure of a proteins (i.e specific a.a. sequence)

Situation #1 – Lactose IS present

When Lactose is digested, it is broken down as follows: beta-galactosidaseLactose ---------------------- glucose & galactose (major) Lactose is known as an inducer – a compound that evokes synthesis of an enzyme - in this example, lactose will induce the production of enzymes Z, Y, and A

1. lactose (inducer) will bind to repressor & cause a shape change

2. Repressor can no longer recognize the operator binding site; switch is turned ON

3. RNA Polymerse can bind to the operon’s promoter

4. RNA Polymerase begins to transcribe the genes & genes are then translated

5. The genes produce enzymes (Z, Y, A) that help break down lactose

1. In the absence of lactose, there is no lactose to bind to the Repressor enzyme & block it from binding to operator2. Thus, Repressor does bind to the operator and the switch is left OFF

Situation #2: Lactose is NOT present

3. RNA Polymerase is now blocked from binding to the lacPromoter

4. Thus NO TRANSCRIPTION of the genes

http://bcs.whfreeman.com/thelifewire/content/chp13/1302001.html

The Trp Operon: A Repressible System

Sometimes tryptophan is present in high concentrations (uh, THANKSGIVING, YUM!!!!!!) so it is advantageous to stop making enzymes for tryptophan synthesis

These enzymes are said to be repressible

Situation #1: Tryptophan is NOT present

1. Repressor gene produces an inactive repressor which cannot bind to operator (so stays ON)2. RNA polymerase can bind to operator, transcribe genes enzymes will make Trp.

Situation #2: Tryptophan IS present

1. Tryptophan (i.e. co-repressor) will bind to repressor protein and activates the repressor

2. Repressor binds to operator 3. RNA polymerase can’t bind to operator, thus genes are not transcribed, no Trp made

http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html

SO, What is the difference b/n Inducible & Repressible Systems? Summarize.

In inducible systems, a substance in the env’t(i.e. the inducer) interacts with the repressor making it incapable of binding to operator andblocking transcription. (enzyme will be produced)

In Repressible systems, a substance in env’t(i.e the corepressor) binds to repressor to makeit capable of binding to operator and blockingtranscription

C. Eukaryotic Gene Regulation/Expression

--Eukaryotes do not have a universal mechanism (i.e operons) that controls the activity of coding genes

--Rather, regulation is possible at any point in the pathway b/n gene to functional protein