RNA and Protein Synthesis

How does DNA “code”?

DNA inherited by an organismLeads to specific traits by dictating

synthesis of proteins DNA directs protein synthesis/gene

expression2 stages - transcription and translation

Central Dogma

DNA RNA PROTEIN

History of RNA Synthesis George Beadle & Edward Tatum in

(‘40s)- ‘One gene, one enzyme’ Function of a gene is to dictate the

production of a specific enzyme

Beadle

1903-1989

American

Nobel Prize 1958

Tatum

1909-1975

American

Nobel Prize 1958

After Beadle & Tatum Some genes encode proteins that

are not enzymes One gene is responsible for one

polypeptide chain, and some proteins have more than one chain

One gene, one polypeptide hypothesis

The BIG Picture DNA is transcribed to form RNA RNA is translated to form protein

Prokaryotic cell - no nucleus, mRNA produced by transcription immediately translated without additional processing

TRANSLATION

TRANSCRIPTION DNA

mRNA

Ribosome

Polypeptide

Eukaryotic cell - nucleus provides a separate compartment for transcription. Original RNAtranscript (pre-mRNA) processed in various ways before leaving nucleus as mRNA

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

mRNA

DNA

Pre-mRNA

Polypeptide

Ribosome

Nuclearenvelope

RNA RNA is used as an intermediary

between DNA and proteins RNA is a single strand nucleotide

polymer Composition

Sugar- RibosePhosphate group(s)Uracil substitutes for thymine

uracil

Transcription Overview A copy of the DNA is

made in the form of mRNA (messenger RNA) in transcription

Translation Overview Translation involves mRNA, tRNA

(transfer RNA), and rRNA (ribosomal RNA) coordinating to produce proteins

Translation Overview 2 mRNA has sequences of 3

nucleotides called codons Codons are read in sequences of 3

called triplet code

Translation Overview 3

Codons are written 5’ to 3’ fashion

Each codon codes for one amino acid

Codons do not overlap

Translation Overview 4 Four bases can combine in 43

combinations– more than enough to code for the 20 naturally occurring amino acids

43 = 64 Why don’t we have 64 amino acids?

Universal Code Genetic code (AA

code) nearly universalFrom simplest bacteria

to complex animals Genes can be

transcribed & translated after being transplanted from one species to another

Tobacco plant with firefly gene

Translation Overview - tRNA tRNA molecule has a

sequence of 3 nucleotides- the anticodon

Anticodons base pair with the codon in a complementary way

Anticodons are written in 3’ to 5’ direction

Translation Overview 6 Ribosomes are composed of proteins

and rRNA

The E, P, and A are rRNA

Ribosome

Transcription: Initiation

Synthesis of RNA from the DNA template

Main enzyme is RNA polymerase Transcription does not involve a

primer - it begins at a promoter site The promoter is a “start” sequence

Transcription: Elongation RNA synthesis proceeds in a 5’-3’

direction copying DNA from the 3’-5’

Upstream- towards 5’ end of mRNA sequence

Downstream- towards the 3’ end of mRNA sequence

Transcription: Elongation

RNA polymerase moves along the DNA

Untwists the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Transcription: Elongation Bacterial promoters are about 40

bases long and are located in the DNA just upstream from the starting point

Transcription: Termination Sequences at the end of the gene act as

stop signals Typically only one strand of DNA is

transcribed and is called the template strand

Termination (eukaryotes) Enzymes in nucleus modify pre-mRNA

(before genetic messages sent to the cytoplasm)

mRNA contains additional base sequences that do not directly code for proteins5’ end: modified nucleotide cap3’ end: poly-A tail

`

5’ Cap & Poly A Tail

5’ CapProtect mRNA from hydrolytic enzymesFunctions as an “attach here” signal for

ribosomes Poly A Tail

50 – 250 nucleotidesSame function as 5’ capFaciliates export from nucleus

RNA Modification

RNA splicingRemoves introns and joins exonsIntrons – noncoding regions of DNAExons – coding portions of DNA Introns stay “in” the nucleus, exons

“exit” the nucleus

Spliceosome

Splicing accomplished by a spliceosomeConsists of a variety of proteins and

several small nuclear ribonucleoproteins (snRNPs)

Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA)

Each is about 150 nucleotides long

Translation 1 During translation, the nucleic acid

message is decoded An amino acid is attached to tRNA

before becoming incorporated into a polypeptideTo form a polypeptide chain, the amino

and carboxyl groups of amino acids are joined

Translation 2

The specific sequence of the amino acids (primary structure) is dictated by the sequence of codons of the mRNA

Translation 3

tRNA is linked to amino acids by aminoacyl-tRNA synthetases

This is an energy requiring process

RNA molecules - tRNA

RNA molecules have specialized regions with specific functions

tRNA molecules have attachment sites for amino acids

RNA molecules

tRNA molecules have anticodons that bind to complementary codons of the mRNA

If the mRNA codon is UAC, then what is the anticodon present on the tRNA?

RNA molecules tRNA must be recognized by both the

specific aminoacyl-tRNA synthetase and the ribosome

RNA molecules tRNA ~ 70 nucleotides

long, some generic sections & some unique sections

The nucleotide chain is folded back upon itself to form 3 or more loops with unpaired nucleotides exposed

Ribosomes Components of

translational machinery come together at the ribosomes

Ribosomes are composed of two subunits

Ribosomes The large subunit has a

groove into which the small subunit fits

Ribosomes are transcribed from DNA, but do not carry information

Function as physical site of translation & as a catalyst

Ribosome The A site of the

ribosome is where the aminoacyl-tRNA binds

The P site is where the tRNA holding the polypeptide chain is positioned

Translation Steps Inititiation Elongation Termination

Initiation Initiation factors

(proteins) move an initiation tRNA onto the small ribosomal subunit

The codon for the initiation is AUG, which codes for the amino acid methionine

Initiation 2 Initiation

complex binds to ribosome recognition sequences on the mRNA, and aligns anticodon of tRNA with the codon of mRNA

Initiation 3 The large

ribosomal subunit then binds, forming the functional ribosome

Elongation 1 Addition of new

amino acids Initiator tRNA is

bound to P site of the ribosome - A site is unoccupied until the next aminoacyl-tRNA moves in

Elongation 2 Energy for this

process comes from GTP

Peptide bond formation

Amino group of the new amino acid & carboxyl group of “old” amino acid

Elongation 3 Protein synthesis proceeds from the

amino end to the carboxyl end The tRNA molecule is released from

the P site requiring ribozyme, peptidyl transferase

Translocation - movement of the growing polypeptide chain from the A site to P site - energy comes from GTP

Elongation 4 Translation of the mRNA proceeds in

a 3’ to a 5’ direction, which is the same as the direction of transcription

Translocation ensures

Termination Occurs when the mRNA presents the

codons UAA, UGA, or UAG; No complementary tRNA Release factors recognize codons The ribosome dissociates into the

two subunits

Translation Tutorial http://telstar.ote.cmu.edu/Hughes/Hu

ghesArchive/tutorial/polypeptide/tutorial.swf

Polyribosome A single mRNA can make many copies

of a polypeptide simultaneously Multiple ribosomes, polyribosomes,

may trail along the same mRNA

Mutations Point mutations – single bp change Inheritable (if occurs in gametes)

Mutations are changes in DNA

Point mutation

Base pair substitutionReplacement of a pair of

complementary nucleotides with another nucleotide pair

Some have little/no impact Silent mutation AA change

SimilarNonessential

Missense mutation

AA change

Nonsense mutation

AA change to stop codon

Mutations Frameshift mutations- involve the

insertion or deletion of a base They result in an entirely different

sequence of amino acids because they change the _______________

Mutations are changes in DNA

Frameshift Mutations

Mutations Transposons - movable sequences

of DNA that may move into another area of DNAMay disrupt genes, but may inactivate

othersTransposons have some similarities to

retroviruses

Mutations are changes in DNA

Transposons

Barbara McClintock (1902-1992)

Nobel Prize 1983 Cornell University Discovered the

transposons “jumping genes”

Studied maize

Mutations

Hot spots - regions of DNA more likely to undergo mutations, and are often regions of repeated nucleotides, causing the polymerases to slip

Mutagens Agents that cause mutations

Including ionizing radiationMutations in somatic cells are not

passed on to the next generationSome mutagens are also carcinogens