Transcription and translation

The link between genes and enzymes

“Inborn errors of metabolism”

• A. Garrod, 1902– Certain diseases seemed to be inherited– Alkaptonuria due to an enzymatic defect

• Beadle and Tatum, 1941- one gene, one enzyme– Studied Neurospora crassa– Nutritional (auxotrophic) mutants– Biosynthetic pathways

Beadle and Tatum experiment• Pathway for arginine

biosynthesis was known• Used media with

defined supplements to identify mutants

• One gene, one polypeptide hypothesis defines relationship between DNA and protein

The central dogma of molecular biology

DNARNAProtein

RNA is involved in protein synthesis, too

• Messenger RNA- the gene sequence• Ribosomal RNA- structural component of the

ribosome• Transfer RNA- interpret mRNA and build the amino

acid sequence on the ribosome• In eukaryotes:– Small nuclear RNAs-– SRPs (signal recognition particles)– miRNA (micro-RNA)

The triplet code

• Four nucleotides• Triplets give 64 possible

combinations to code for 20 amino acids

• Code is nonoverlapping• One DNA strand serves

as the template

Transcription: RNA copied from DNA

• RNA polymerase uses DNA as a template to make RNA

• Template is the “antisense” strand5’-TACGGTACATTCGTACC ATC T -3’3’-ATGCCATGTAAGCATGGTAGA -5’

mRNA:5’- UACGGUACAUUCGUACCAUCU-3’

Deciphering the genetic code• 64 possible codons• Experimental requirements:– Cell-free systems with all the necessary enzymes– Synthetic RNAs

• Nirenberg, 1961-6:– Worked with different combinations of RNAs to deduce

codons• Khorana (overlapped Nirenberg)

– Synthesized RNA molecules of defined sequences and analyzed peptides produced

– Also synthesized acetyl CoA and the first artificial chromosome (1970)

Some exceptions to the code

• Genetic code is shared by all organisms• Some variations in mitochondrial, chloroplast

and protozoan DNA (ribosomes are different, too)

• Processes of transcription and translation are very similar in prokaryotes and eukaryotes, but not identical

• Initiation, elongation, termination

Properties of RNA polymerase

• Two forms: holoenzyme and core polymerase• Need both for accurate initiation of RNA

synthesis• Promoter and start site• Accurate termination• Process is simpler in prokaryotes: one

polymerase

Models of transcription events

Operons: transcription and translation coupled in prokaryotes

Regulatory promoter operator structural genesregion

repressor

Eukaryotic vs prokaryotic transcription

• Eukaryotes have three RNA polymerases, each with their own promoters– RNA polymerase I- rRNA• Probably species-specific

– RNA polymerase II- mRNA and snRNAs• Core promoter

– RNA polymerase III- tRNA and some small RNAs• Promoter is internal

– All work in cell nucleus

Eukaryotic initiation complex contains many cofactors

Note that transcription factors bind first

Elongation and termination of transcription

• Multiple molecules of polymerase can transcribe DNA simultaneously

• Termination mechanism is different in eukaryotes

Another difference: posttranscriptional modification

• 5’ cap– Guanine is methylated

and linked to 5’ end of transcript

• 3’ poly-A tail– Specific cleavage site

AAUAA– Poly-A polymerase adds

poly-A tail after that site

• Splicing of “pre-mRNA”

Eukaryotic mRNA is spliced

• Introns and exons• Much more human DNA

is in introns than exons (exons are 1-1.5% of total)

• Splicing involves snRNPs

Model for pre-mRNA splicing

What are the rules for intron frequency and size?

• There are none• Some genes have many introns, some none• Probably accumulated over time• “Exon shuffling” seen among some families of

proteins• Alternative splicing seen (remember the

codon rule)

Overview of translation

Structure of transfer RNA (tRNA)

tRNA charging ensures that amino acids are positioned correctly

tRNA and the ribosome• E site- for tRNA with

amino acid already added (exit)

• P site- for amino acid being added (peptidyl)

• A site for the incoming amino acid (aminoacyl)

• This is where peptide bonds are formed (remember primary structure?)

Initiation of translation in prokaryotes:note the order of events

AUG is start codon; first amino acid is N-formylmethionineMethionine in eukaryotes; also more initiation factorsSmall subunit binds to 5’-cap instead of a ribosome-binding sequence

Elongation cycle

Wobble pairinggives flexibility(note orientation of anticodon)

Termination triggered by a stop codon

In eukaryotes, many proteins are processed in the ER

• Prokaryotes have this mechanism, too

• Secreted proteins require it

Summary: gene expression in bacteria

Gene expression in eukaryotes

Mutations occur in DNA

Does mutation affect phenotype?

• Triplet repeats- add to reading frame but do not shorten it

• Might be in coding or noncoding region

• Sometimes they are big enough to see in a karyotype

Chromosomal mutations can have drastic effects

• Deletions• Duplications

– Can lead to “gene families” and pseudogenes

• Inversions• Translocations• Can be fatal, can be

inherited; tend to arise in single cells (somatic mutation)

Summary• Genes specify polypeptides• Transcription makes RNA copies of DNA• Translation involves mRNA, tRNA, and rRNA in

protein synthesis• Processes are similar in prokaryotes and eukaryotes,

but there are significant differences• The genetic code is nearly universal• Mutations alter DNA and can alter genes and

proteins• Evolution arises from mutation