Gene Expression : Transcription and Translation

How Are Different Types of Cells Created and Maintained? By differential gene expression. The same genetic information is in all 100 trillion cells of any one person. Different cells use the same blueprint in different ways. How? In essence, the control of gene expression occurs by regulating the flow of information from DNA to protein.

The “Central Dogma” of Molecular Genetics Transcription Translation DNA RNA Protein Trait RNA processing

Review DNA RNA Proteins Made up of 4 different nucleotides Made up of 20 different amino acids

Gene Expression in Prokaryotes vs. Eukaryotes Transcription and translation are separated in time and space Transcription and translation may occur simultaneously

One Gene - One Polypeptide Hypothesis Theory: one gene codes for one polypeptide Some proteins are composed of a number of polypeptide chains. In this theory each chain has its own gene. However, eukaryotic genes are much more complex and this is not always the case! Some genes control the expression of other genes Some genes code for RNA which do not produce polypeptides

The Genetic Code Problem: How do only 4 different nucleotides code for the 20 amino acids that make up proteins? Solution: Each group of 3 nucleotides codes for a different amino acid. These 3 nuclotide units are called codons. 1 codon codes for 1 amino acid. DNA RNA Amino Acid C G Valine A U T A

Reading Frame The 3-nucleotide units (codons) must be read in the correct reading frame Start codons determine the reading frame

The Genetic Code Dictionary There are multiple codons for each amino acid AUG is always the start codon UAA, UGA, and UAG are stop codons

Characteristics of the Genetic Code The genetic code is degenerate More than one codon codes for an amino acid The genetic code is universal (almost) Almost all organisms on Earth use the same code.

Transcription DNA is used to make a strand of RNA called the primary transcript (pre-mRNA) The pre-mRNA is further processed to create the finished mRNA mRNA exits the nucleus to be translated

DNA has two strands 1) Anti-sense strand (Template strand) (Runs 3’ 5’) – the strand of DNA that is transcribed. Has the complementary genetic code of the mRNA. (Runs 3’ 5’)  2) Sense strand (coding strand) (runs 5’  3’) – the strand of DNA that is not transcribed. It has the same genetic code as the mRNA (except U instead of T).

Transcription 3 main steps 1. Initiation 2. Elongation 3. Termination

Transcription Initiation RNA polymerase binds to DNA at a region called the promoter RNA polymerase unwinds the DNA and adds nucleotides in the 5’ → 3’ direction

Transcription Elongation RNA polymerase moves along the DNA strand, adding 60 nucleotides/sec DNA strands rejoin after polymerase passes by

Transcription Termination Polymerase stops when it reaches a DNA sequence called the terminator The mRNA has been completely transcribed In eukaryotes, this is pre-mRNA and must be further processed

mRNA Processing In eukaryotes, pre-mRNA must be further processed to mRNA before it leaves the nucleus Guanine is added to 5’ end, forming the 5’ cap 100’s of adenines are added to 3’ end, forming the poly-A tail Non-coding regions of RNA are spliced out

Intron (non-coding sequences) are cut out by spliceosomes Intron (non-coding sequences) are cut out by spliceosomes. Leaving only Exons (Coding sequences) making up the mRNA that leaves the nucleus. Alternative splicing patterns means one gene can make more than one protein

mRNA Splicing

Translation The process in which mRNA is used to make proteins Occurs in the cytoplasm using ribosomes Requires tRNA (transfer RNA) bound to an amino acid 3 steps: initiation, elongation, termination

Structure of tRNA A clover-shaped RNA molecule Bottom loop has an anti-codon complementary to the mRNA codon 3’ end has an aa attachment site with the sequence “ACC” (CCA read from 5’ 3’)

Amino acid attachment RNA is made in the nucleus Amino acids float free in the cytoplasm Aminoacyl-tRNA synthase joins each amino acid to the appropriate tRNA

Ribosomes 2 subunits Composed of proteins and rRNA 3 tRNA binding sites

Translation: Initiation mRNA, tRNA and small ribosomal subunit bind with the P site at the start codon (AUG = Met) Large subunit binds using energy from GTP

Translation: Elongation mRNA is read 3 nucleotides at a time (Codons) tRNA brings corresponding amino acid into the A site of the ribosome

Transition Elongation Ribosome catalyses dehydration synthesis reaction between aa’s in P site and A site forming a peptide bond between aa’s Growing polypetpide now attached to tRNA in A site Ribosome moves forward one codon Free tRNA in P site exits out the back of ribosome (out of E site) tRNA (with polypeptide) moves into P site

Translation: Termination Elongation continues until reaching a stop codon Release factor binds and hydrolyzes the bond between the last tRNA and its a.a., freeing the new polypeptide chain

Polyribosomes Many ribosomes may simultaneously translate from a single mRNA

Gene Expression: Overview