Protein Synthesis

• Genome - the genetic information of an organism

• DNA – in most organisms carries the genes

• RNA – in some things, for example retroviruses like the AIDS virus

• Gene - a DNA sequence that is transcribed (includes genes that do not encode proteins)

Information specifying protein structure

• Information flow: CENTRAL DOGMA

• DNA RNA PROTEIN

• Transcription - copying of the DNA sequence information into RNA

• Translation - Information in RNA molecules is translated during polypeptide chain synthesis

Biological information flow

Types of RNA

(1) Transfer RNA (tRNA)•Carries amino acids to translation

machinery•Very stable molecules

(2) Ribosomal RNA (rRNA) •Makes up much of the ribosome•Very stable, majority of cellular RNA

(3) Messenger RNA (mRNA) •Encodes message from DNA to ribosomes•Rapidly degraded by nucleases

Biological information flow

RNA Polymerase

• RNA polymerase (RNA pol) catalyzes DNA-directed RNA synthesis (transcription)

• RNA pol is core of a larger transcription complex

• Complex assembles at one end of a gene when transcription is initiated

• DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain

The Chain Elongation Reaction

• Mechanism almost identical to that for DNA polymerase

• Growing RNA chain is base-paired to DNA template strand

• Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template

• New phosphodiester bond formed, PPi released

RNA polymerase reaction

• Catalyzes polymerization in 5’ 3’ direction

• Is highly processive, and thermodynamically assisted by PPi hydrolysis

• Incoming RTPs: UTP, GTP, ATP, CTP

• Growing single-stranded RNA released

• Adds 30-85 nucleotides/sec (~ 1/10th rate of DNA replication)

• RNA polymerase reaction

Transcription Initiation

• Transcription complex assembles at an initiation site (DNA promoter region)

• Short stretch of RNA is synthesized

• Operon: a transcription unit in which several genes are often cotranscribed in prokaryotes

• Eukaryotic genes each have their own promoter

Transcription of E. coli ribosomal RNA genes

A. Genes have a 5’ 3’ Orientation

• Convention for double-stranded DNA:Coding strand (top) is written: 5’ 3’Template strand (bottom) is written: 3’ 5’

• Gene is transcribed from 5’ end to the 3’ end

• Template strand of DNA is copied from the 3’ end to the 5’ end

• Growth of RNA chain proceeds 5’ 3’

Orientation of a gene

Transcription Complex Assembles at a Promoter

• Consensus sequences are found upstream from transcription start sites

• DNA-binding proteins bind to promoter sequences (prokaryotes and eukaryotes) and direct RNA pol to the promoter site

Promoter sequences from 10 bacteriophage and bacterial genes

E. coli promoter

(1) TATA box (-10 bp upstream from transcription start site (rich in A/T bp)

(2) -35 region (-35 bp upstream) from start site

• Strong promoters match consensus sequence closely (operons transcribed efficiently)

• Weak promoters match consensus sequences poorly (operons transcribed infrequently)

Initiation of transcription in E. coli(two slides)

Transcription Termination

• Only certain regions of DNA are transcribed

• Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences

Transcription in Eukaryotes

A. Eukaryotic RNA Polymerases

• Three different RNA polymerases transcribe nuclear genes

• Other RNA polymerases found in mitochondria and chloroplasts

• Table 21.4 (next slide) summarizes these RNA polymerases

Eukaryotic Transcription Factors

• Same reactions as prokaryotic transcription

• More complicated assembly of machinery

• Binding of RNA polymerase to promoters requires a number of initiation transcription factors (TFs)

Transcription of Genes Is Regulated

• Expression of housekeeping genes is constitutive

• These genes usually have strong promoters and are efficiently and continuously transcribed

• Housekeeping genes whose products are required at low levels have weak promoters and are infrequently transcribed

• Regulated genes are expressed at different levels under different conditions

Role of regulatory proteins in transcription initiation

• Regulatory proteins bind to specific DNA sequences and control initiation of transcription

• Repressors - regulatory proteins that prevent transcription of a negatively regulated gene

• Negatively regulated genes can only be transcribed in the absence of the repressor

• Activators - regulatory proteins that activate transcription of a positively regulated gene

Inducers and corepressors

• Repressors and activators are often allosteric proteins modified by ligand binding

• Inducers - ligands that bind to and inactivate repressors

• Corepressors - ligands that bind to and activate repressors

• Four general strategies for regulating transcription

• Strategies for regulating transcription initiation by regulatory proteins

Posttranscriptional Modification of RNA

• Mature rRNA molecules are generated in both prokaryotes and eukaryotes by processing the primary transcripts

• In prokaryotes, 1o transcripts often contain several tRNA precursors

• Ribonucleases (RNases) cleave the large primary transcripts to their mature lengths

Ribosomal RNA Processing

• Ribosomal RNA in all organisms are produced as large primary transcripts that require processing

• Processing includes methylation and cleavage by endonucleases

• Prokaryotic rRNA primary transcripts ~30S

• Contain one copy each: 16S, 23S, 5S rRNA

Eukaryotic mRNA Processing

• In prokaryotes the primary mRNA transcript is translated directly

• In eukaryotes transcription occurs in the nucleus, translation in the cytoplasm

• Eukaryotic mRNA is processed in the nucleus without interfering with translation

• In some mRNA, pieces are removed from the middle and the ends joined (splicing)

Eukaryotic mRNA Molecules Have Modified Ends

• All eukaryotic mRNA precursors undergo modifications to increase their stability and make them better substrates for translation

• Ends are modified so they are no longer susceptible to exonuclease degradation

• The 5’ ends are modified before the mRNA precursors are completely synthesized

• Guanylate base is methylated at N-7

• 2- Hydroxyl groups of last two riboses may also be methylated

Poly A tails at the 3’ ends of mRNA precursors

• Eukaryotic mRNA precursors are also modified at their 3’ ends

• A poly A polymerase adds up to 250 adenylate residues to the 3’ end of the mRNA precursor

• This poly A tail is progressively shortened by 3’ exonucleases

• The poly A tail increases the time required for nucleases to reach the coding region

Some Eukaryotic mRNA Precursors are Spliced

• Introns - internal sequences that are removed from the primary RNA transcript

• Exons - sequences that are present in the primary transcript and the mature mRNA

• Splice sites - junctions of the introns and exons where mRNA precursor is cut and joined

Triose phosphate isomerase gene (nine exons and eight

introns)