PROTEIN SYNTHESIS

dr. Isra Thristy, M.Biomed

Genes and Regulatory Elements

• Structural genes: encoding proteins

• Regulatory genes: encoding products that interact with other

sequences and affect the transcription and translation of these
sequences

• Regulatory elements: DNA sequences that are not transcribed but play
a role in regulating other nucleotide sequences
Genes and Regulatory Elements

■ Constitutive expression: continuously expressed under normal cellular

conditions

■ Positive control: stimulate gene expression

■ Negative control: inhibit gene expression

Level of Gene Regulation

Many levels at which gene expression may

be regulated
• DNA access
• Transcription
• RNA modification and stability
• Translation
• Post-translational modification
DNA-Binding Proteins

• Domains: 60 ~ 90 amino acids, responsible for binding to DNA, forming

hydrogen bonds with DNA

• Distinctive types of DNA-binding proteins based on the motif

• Motif: within the binding domain, a simple structure that fits into
the major groove of the DNA
Transcription in Eukaryotes

■ Three RNA polymerases are known; each transcribes a different set

of genes and recognizes a different set of promoters:
• RNA Polymerase I- found in the nucleolus and synthesizes
precursors of most rRNAs
• RNA Polymerase II- found in the nucleoplasm and synthesizes
mRNA precursors
• RNA Polymerase III- found in the nucleoplasm and synthesizes
tRNAs, other RNA molecules involved in mRNA processing and
protein transport
How does Pol II Recognize the Correct
DNA?
■ Four elements of the Pol II promoter allow for this phenomenon
Initiation of Transcription

■ Any protein regulator of transcription that is not itself a subunit of Pol

II is a transcription factor

■ Initiation begins by forming the preinitiation complex

■ Transcription control is based here

General Transcription Initiation Factors
Elongation and Termination

■ Elongation is controlled by:

– pause sites, where RNA Pol will hesitate
– anti-termination, which proceeds past the normal termination
point
– positive transcription elongation factor (P-TEF) and negative
transcription elongation factor (N-TEF)
■ Termination
– begins by stopping RNA Pol; the eukaryotic consensus sequence
for termination is AAUAAA
Gene Regulation
■ Enhancers and silencers- regulatory sequences that augment or
diminish transcription, respectively
■ DNA looping brings enhancers into contact with transcription
factors and polymerase
Eukaryotic Gene Regulation
■ Response elements are enhancers that respond to certain metabolic
factors
• heat shock element (HSE)
• glucocorticoid response element (GRE)
• metal response element (MRE)
• cyclic-AMP response element (CRE)

■ Response elements all bind proteins (transcription factors) that are

produced under certain cell conditions
Response Elements
Activation of transcription Via CREB and CBP

■ Unphosphorylated CREB
does not bind to CREB
binding protein, and no
transcription occurs
■ Phosphorylation of CREB
causes binding of CREB to
CBP
■ Complex with basal
complex (RNA polymerase
and GTFs) activates
transcription
Structural Motifs in DNA-Binding
Proteins
■ Most proteins that activate or inhibit
RNA Pol II have two functional domains:
– DNA-binding domain
– transcription-activation domain
■ DNA-Binding domains have domains
that are either:
• Helix-Turn-Helix (HTH)
• Zinc fingers
• Basic-region leucine zipper
Helix-Turn-Helix Motif
Hydrogen bonding between amino acids and DNA
Zinc Finger Motif
■ Motif contains 2 cysteines and 2 His
12 amino acids later
■ Zinc binds to the repeats
Basic Region Leucine Zipper Motif
■ Many transcription factors contain this motif, such as CREB
(Biochemical Connections, page 315)

■ Half of the protein composed of basic region of conserved Lys, Arg,

and His

■ Half contains series of Leu

■ Leu line up on one side, forming hydrophobic pocket

Helical Wheel Structure of Leucine Zipper
Transcription Activation Domains

– acidic domains- rich in Asp and Glu. Gal4 has domain of 49

amino acids, 11 are acidic

– glutamine-rich domains- Seen in several transcription factors.

Sp1 has 2 glutamine-rich domains, one with 39 Glu in 143 amino
acids

– proline-rich domains- Seen in CTF-1 (an activator). It has 84

amino acid domain, of which 19 are Pro
Post Transcriptional RNA Modification
■ tRNA, rRNA, and mRNA are all modified after transcription to give the
functional form
– the initial size of the RNA transcript is greater than the final size
because of the leader sequences at the 5’ end and the trailer
sequences at the 3’ end
– the types of processing in prokaryotes can differ greatly from that
in eukaryotes, especially for mRNA
■ Modifications
– trimming of leader and trailer sequences
– addition of terminal sequences (after transcription)
– modification of the structure of specific bases (particularly in
tRNA)
Posttranscriptional Modification of tRNA
Precursor
Modification of tRNA
■ Transfer RNA- the precursor of
several tRNAs is can be
transcribed as one long
polynucleotide sequence
– trimming, addition of
terminal sequences, and
base modification all take
place
– methylation and
substitution of sulfur for
oxygen are the two most
usual types of base
modification
Modification of rRNA
■ Ribosomal RNA
– processing of rRNA is primarily a matter of methylation and
trimming to the proper size

– in prokaryotes, 3 rRNAs in one intact ribosome

– in Eukaryotes, ribosomes have 80s, 60s, and 40s subunits

– base modification in both prokaryotes and eukaryotes is primarily

by methylation
Modification of mRNA
– Includes the capping of
the 5’ end with an N-
methylated guanine that
is bonded to the next
residue by a 5’ -> 5’
triphosphate.

– Also, 2’-O-methylation of
terminal ribose(s)
mRNA Modification
– A polyadenylate “tail” that is usually100-200 nucleotides long, is
added to the 3’ end before the mRNA leaves the nucleus
– This tail protects the mRNA from nucleases and phosphatases
– Eukaryote genes frequently contain intervening base sequences
that do not appear in the final mRNA of that gene product
– Expressed DNA sequences are called exons
– Intervening DNA sequences that are not expressed are called
introns
– These genes are often referred to as split genes
Organization of Split Genes in Eukaryotes
The Splicing Reaction
■ Exons are separated by
intervening intron

■ When the exons are

spliced together,a lariat
forms in the intron
Ribozymes
■ The first ribozymes discovered included those that catalyze their own
self-splicing
■ More recently, ribozymes have been discovered that are involved in
protein synthesis
■ Group I ribozymes
– require an external guanosine
– example: pre-rRNA of the protozoan Tetrahymena (next screen)
■ Group II ribozymes
– display a lariat mechanism similar to mRNA splicing
– no requirement for an external nucleotide
Self-splicing of pre-rRNA