D1.2: Protein Synthesis

Transcription

synthesis of an RNA molecule from a DNA template using RNA Polymerase II

Purpose of Using mRNA (2)

Protection and Speed

Protection

DNA is kept safely in nucleus

Speed

one gene can be converted to many mRNAs, one mRNA can be converted to many polypeptides

Therefore speeds up process

RNA Polymerase II

transcribes mRNA

RNA Polymerase II Process

1. binds to promoter (DNA sequence) to begin transcription

2. unwinds/separates DNA

3. Moves along template DNA, produces single mRNA strand, complementary to DNA by joining ribonucleoside triphosphates (rNTPs) covalently, growing in the 5' --> 3' direction

4. Detaches at terminator and allows DNA to reform double helix, releases mRNA which eventually leaves via nuclear pores

Complementary Base Pairing

Each RNA nucleotide added is complementary to the corresponding base on template DNA (A-U, C-G)

*cannot form h-bonds w/ other bases*

Sense vs Antisense Strand

Either of two DNA strands may contain the gene of interest, so the designation varies

Sense Strand

The coding strand (NONTEMPLATE), contains the gene to be transcribed into mRNA and identical to the mRNA but w/ T instead of U

Antisense Strand

The TEMPLATE strand (noncoding), contains the complementary base sequences to the mRNA

This is the actual strand that RNA Polymerase II moves along, and is the location of the promoter and the terminator

Stability of DNA

Stability is essential bc DNA is transcribed many times during a cell's life, therefore transcription should not impact DNA

Once transcription completed, 2 DNA strands close again by reforming hydrogen bonds to complementary bases

DNA only vulnerable to chemical changes (mutation) when RNA Polymerase II moves along DNA

**if mutations were common, mRNA would contain increasing amounts of errors*

Gene Expression

Process in which information in genes has observable effects on organisms

Function of most genes is to produce AAs in a polypeptide, which result in proteins that determine observable traits

Transcription is the first stage of gene expression where genes can be switched on or off

Variability of Expressed Genes

Some genes are always transcribed bc their proteins are required (housekeeping genes)

Some genes are never expressed in the life of a cell

ex. insulin gene in a skin cell

Housekeeping Genes

a gene that is transcribed continually because its product is needed at all times and in all cells

Transcriptome

all the RNA molecules transcribed from a genome

Transcription Initiation

RNA polymerase binds to a promoter

Promoter

DNA sequence that RNA Polymerase II binds to, to begin transcription

Found upstream of gene of interest on template strand, regulated via transcription factors and regulatory proteins

Transcription Factors

Group of proteins that guide RNA Polymerase II to the promoter, making it easier to bind

Can change gene expression

RNA Polymerase II usually cannot begin transcription without them

Regulatory Proteins

Bind to DNA upstream of promoter and affects transcription factors

Either activators or repressors

Activators

Proteins that encourage transcription factors and RNA Polymerase II to bind to DNA, increases transcription

Repressors

Proteins that prevent transcription factors and RNA Polymerase II from binding, decreases transcription

Control Elements

DNA sequences that regulatory proteins bind to (most genes have multiple to control gene expression)

Either Proximal elements or Distal elements

Proximal Elements

Located close to promoter, transcription factors typically bind here

Distal Elements

Located farther upstream from promoter, activators and repressors typically bind here

Transcription Factors Image

Types of Non-Coding DNA (4)

1. Base sequences that do not code for polypeptides but have other functions like RNA genes that are not translated into proteins (make rRNA/tRNA/snRNA)

2. Introns

3. Regulators of Gene Expression

4. Telomeres

Regulators of Gene Expression

Turns genes on/off by regulating transcription (promotors, proximal elements, distal elements)

Telomeres

Repetitive DNA at the end of linear chromosomes, postpones deterioration that occurs during replication

Why are telomeres necessary?

DNA replication cannot extend all the way to the end of the chromosome and as a result, we lose sequences of information with each round of replication (can't replace RNA primer at end of DNA strand being replicated)

Advantages of Separate Nucleus

Transcription and Translation occur independently (eukaryotes)

Translation happens after mRNA leaves the nucleus

This allows for post-transcriptional modification, which isn't possible in prokaryotes bc translation occurs before transcriptions finishes

Post-Transcriptional Modification

Changes in mRNA after its production, before translation, allows new mRNA to convert into mature mRNA before exiting nucleus

5' Cap

Adds a guanine w/ an extra methyl group to 5' end

Polyadenylation

Adds a poly-A tail (100-200 Adenines) to 3' end

Function of 5' Cap/Polyadenylation

Stabilizes transcript and helps it move out from nucleus

Protects against digestion from exonucleases

Exonucleases

Cut up DNA (typically viral DNA)

Newly transcribed RNA contains alternating...

Exons and Introns

Exons

Coding sequences expressed into amino acids (about 8.8 per gene), fused together to produce final transcript

Exons Exit nucleus

Introns

intervening, unexpressed sequences (about 7.8 per gene), removed from mRNA and digested into single nucleotides

Introns remain In the nucleus

Alternative Splicing

Allows for one gene to be coded into many different polypeptides, producing polypeptides w/ differing functions w/out need for second, similar gene

Most frequent change is exon skipping

Exon Skipping

when an exon is spliced out of a pre-mRNA

ex. Exon 1, Exon 2, Exon 3 in one mRNA and Exon 1, Exon 3, Exon 4 in another mRNA

Central Dogma

DNA -> RNA -> Protein

Translation

mRNA--> amino acid sequence of proteins

mRNA holds codes necessary for polypeptides, copied from a gene via transcription and translated by ribosomes in cytoplasm

Direction of Transcription/Translation

Enzymes involved in transcription and translation move in 5'-->3' direction

Transcription Direction

RNA Polymerase II adds phosphate group of RNA nucleotides to ribose sugar of growing mRNA strand

Translation Direction

Ribosomes move from 5'-->3' of mRNA

mRNA

consists of codons that specify for amino acids in a polypeptide AND a ribosome binding site

includes START and STOP codons, where translation begins and ends

Can be used multiple time unless damaged or no longer needed

tRNA

Matches codon in mRNA w/ proper amino acid, has anticodon at one end and amino acid attachment site at the other (3')

Each tRNA has a distinctive shape that can be recognized by aminoacyl tRNA synthetase (activating enzyme), that attaches the correct AA to tRNA

Ribosomes

Composed of protein and rRNA (ribozyme)

2 subunits: large and small

Size: 70S in prokaryotes and 80S in eukaryotes

Large Ribosome Subunit

Has A (aminoacyl, 3' end), P (peptidyl), and E (exit, 5' end) sites

Small Ribosome Subunit

mRNA binding site

Adaptation of ribosomes

Large (30 nm) structure of rRNA and proteins

Either free or bound

Free Ribosomes

Make polypeptides in cytoplasm that are either utilized there or enter the nucleus (histones in octamer)

Produce proteins means to be used in cell, including for housekeeping (glycolysis)

Bound Ribosomes

Make polypeptides that must be transported somewhere else in cell or secreted

Placed into lumen (inside space) of rER, transported in vesicles that bud off

Genetic Code

Set of rules by which mRNA is converted to AA sequences in polypeptides

mRNA contains CODONS

Codons

*non-overlapping, triplet bases that code for one AA*

64 possibilities (4^3), codes for 20 AAs

Start Codon

AUG, codes for methionine and begins translation

Stop Codon

UAA, UGA, UAG, codes for release factor which releases polypeptide chain from ribosome by adding H2O for hydrolysis

Genetic Codes are Degenerate

Means that different codons can code for same AAs (ex. GUU and GUC both code for valine)

But, genetic codes are NOT ambiguous (one codon can't mean two different AAs)

Genetic Code must be read in correct reading frame

Reading Frame

On an mRNA, the triplet grouping of ribonucleotides used by the translation machinery during polypeptide synthesis.

Genetic Code is Universal

Same codon can be translated into same AA in all living organisms and viruses

Therefore must have originated EARLY in life to be present in all modern organisms

ex. Human insulin can be made by bacteria

Translation Initiation

Assembly of Components:

1. Aminoacyl tRNA synthetase attaches methionine to initiator tRNA w/ anticodon UAC (activates tRNA)

2. Initiator tRNA binds to small ribosome subunit forming a Ternary Complex, along w/ AA

3. Ternary Complex binds to 5' end of mRNA

4. Scans along until it finds start codon (AUG), forms hydrogen bonds between codon and anticodon

5. Large subunit sets w/ tRNA at P site forming initiation complex

Ternary Complex

Complex formed by binding of activated initiator tRNA to small ribosome subunit

Initiation Complex

When the large ribosome subunit sets w/ tRNA at P site at the beginning of translation

Translation Elongation

**One cycle adds ONE AA to growing polypeptide chain

1. tRNA w/ complementary anticodon binds to A site (on right, near 3'), pairing w/ mRNA via hydrogen bond

2. AA on tRNA in P site forms a peptide bond TO AA in A site (attaches polypeptide chain to amino acid on A site)

3. tRNA in P site becomes in active, all AA on tRNA in A site

4. Ribosome moves down mRNA, codon by codon (5'-->3')

5. Inactivated tRNA moves to E site and exits, tRNA w/ polypeptide in A site moves to P site

6. Different tRNA entered A site and process repeats

Translation Termination

Disassembly of Components

Elongation continues until stop codon

Release factor enters A site, adds water instead of amino acid, releasing polypeptide and disassembling ribosome

Modification of Polypeptides (5)

1. Remove methionine

2. Change AA's r-chains, including phosphorylation or addition of carbohydrates (to help w/ tertiary bonding)

3. Fold polypeptide to stabilize its tertiary structure, using disulfide bonds

4. Remove part of polypeptide to convert propeptide into mature peptides

5. Combine two or more polypeptides to form quaternary structure, can also add non-polypeptide groups (prosthetic) to quaternary structure to make conjugated proteins

Propeptide

A polypeptide created by a ribosome that has not yet been modified

Pre-proinsulin

Inactive insulin precursor (110 AAs) just formed by ribosome, still contains signal peptide

Proinsulin

Precursor of insulin, formed after protease removes 24 AAs (signal peptide) from n-terminal

Pre-proinsulin to Insulin Conversion

1. Insulin gene produces pre-proinsulin

2. protease removes some AAs (called signal peptide) from n-terminal to form proinsulin

3. Proinsulin folded w/ 3 disulfide bonds to stabilize tertiary structure

4. Other proteases break peptide bonds at 2 points, produces 2 separate chains (1 removed)

5. A-chain and B-chain (2 formed chains) are held together by disulfide bonds made earlier

6. 2 AAs removed from B-chain to yield mature insulin

Recycling Amino Acids (Reasons, 3)

Sustaining a functional proteome requires constant protein breakdown and synthesis bc most proteins have a relatively short life.

Cell's activity changes and protein is no longer necessary (ex moving from 1 stage in cell cycle to the next)

Protein structure is easily affected by free radicals or reactive chemicals, proteins become misfolded or denatured and not functional

Proteasomes

Break down proteins that no longer function

Identifies proteins for destruction by tagging them w/ ubiquitin (small proteins, acts as signals for proteasomes)

Proteasomes unfolds and feeds protein into central chamber, active sites of multiple proteases within proteasome break down proteins into oligopeptides, which move out of proteasome

Oligopeptides further digested into AAs in cytoplasm

Ubiquitin

A protein that attaches itself to faulty or misfolded proteins and thus targets them for destruction by proteasomes

Oligopeptides

Short polypeptides